WATER POLLUTION CONTROL RESEARCH SERIES • 12060 EHT 07/70
Use of Fungi Imperfect!
in
Waste Control
U.S. DEPARTMENT OF THE INTERIOR • FEDERAL WATER QUALITY ADMINISTRATION
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WATER POLLUTION CONTROL RESEARCH SERIES
The Water Pollution Control Research Reports describe the results and
progress in the control and abatement of pollution of our Nation's
Waters. They provide a central source of information on the research,
development and demonstration activities of the Federal Water Quality
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ters as supplies permit. Requests should be sent to the Project Reports
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Federal Water Quality Administration, Washington, D. C. 20242
Previously issued reports on the Food Processing/Industrial Pollution
Control Program:
12060 10/69 Current Practice in Potato Processing
Waste Treatment
12060FAD10/69 Aerobic Treatment of Fruit Processing
Wastes
16080 11/69 Nutrient Removal From Cannery Wastes
By Spray Irrigation of Grassland
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Use of Fungi Imperfect! in Waste Control
by
North Star Research and Development Institute
3100 38th Avenue South
Minneapolis, Minnesota 55406
for the
FEDERAL WATER QUALITY ADMINISTRATION
U.S. DEPARTMENT OF THE INTERIOR
Grant No. 12060 EHT
July 1970
For sale by the Superintendent of Documents, U.S. Government Printing Office
Washington, D.C. 20402 - Price $1
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FWQA Review Notice
This report has been reviewed by the Federal Water
Quality Administration and approved for publication.
Approval does not signify that the contents necessarily
reflect the views and policies of the Federal Water
Quality Administration, nor does mention of trade
names or commercial products constitute endorsement
or recommendation for use.
ii
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ABSTRACT
Forty-five species of twelve genera of the Fungi Imperfect! were
screened for those fungal candidates best able to rapidly convert
soluble and suspended organic material (as measured by BOD) from
corn- and soy food-processing waste streams to mycelial protein.
Rapidly growing fungal strains were selected which were readily
removed from the digested waste effluents by coarse filtration.
Trichoderma viride, Gliocladium deliquescens, and either Asper-
gillus oryzae or (5. deliquescens gave the best results on corn,
soy and SOg-containing soy wheys, respectively. Optimal growth
conditions included pH of 3.2 to 3.5, and a temperature of 30°C.
Oxygen requirements were relatively low (1 Ib Os/6 to 1 Ib COD
removed). Nitrogen and phosphate additions were required for the
corn digestion system, and additions of sulfuric acid were neces-
sary to adjust the pH. These studies were done in 125 ml flasks
containing nonsterile corn and soy wastes. The growth conditions
that resulted in the highest fungal yield and greatest reduction
in BOD and total solids were incorporated into 20-liter continuous
culture digestions. Corn waste was reduced from an initial BOD
level of 1600 mg/1 to 25 mg/1 in 24 hours. Soy wastes were reduced
from 6200 mg of BOD/1 to 125 mg of BOD/1 in 36 hours of incubation.
Studies of rapid fungal digestion of soy whey containing 700 mg/1
of SOs resulted in selection of A. oryzae and G. deliquescens
strains which removed SOg from the medium. Mycelial yields were
approximately 50 to 60 g of dry mycelium per 100 g of COD utilized.
The stability of the continuous fermentation with corn waste was
demonstrated in a fermentation run of 140 days' length. Runs of
30 days' length have been conducted with soy whey. The protein
content of mycelium recovered from the continuous culture corn
digestion system was 45 percent. The recovered mycelium was light
tan in color and bland in taste and smell. Feeding trials in wean-
ling rats using T_. viride grown in corn waste as the protein source
gave a growth response equal to that seen with a standard casein
rat diet. Digestibility was 90 percent, and no toxicity was ob-
served in a three-week trial. Feeding trials were inconclusive
with rats fed G. deliquescens fungal protein from the soy whey
fermentation due to a palatability problem. Economic estimates
based on the experimental results showed the fungal product to be
comparable in cost to soy oil meal.
Results on both soy and corn wastes gave definite encouragement
that the commercial use of selected strains of certain species of
Fungi Imperfecti to remove BOD in a readily harvested form is
practical.
This report was submitted in fulfillment of project 12060 EHT under
the partial sponsorship of the Federal Water Quality Administration.
111
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CONTENTS
Section Page
I. SUMMARY 1
Corn Waste 1
Soy Waste 3
II. INTRODUCTION 5
III. BACKGROUND 7
IV. MATERIALS AND METHODS 9
Fungal Stocks 9
Media 10
Incubation 10
Continuous Culture Apparatus 11
Analytical Measurements 13
Feeding Studies 14
V. RESULTS 17
Corn Waste 17
Soy Whey: HC1 Soy Whey 40
Soy Whey: S02 Soy Whey 59
VI. ECONOMIC ESTIMATES 79
VII. ACKNOWLEDGMENTS 81
VIII. REFERENCES 83
v
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FIGURES
Page
1. Fermentor apparatus for continuous digestion of 12
corn and soy waste by fungi
2. Strain selection of fungi for rapid COD reduction 19
on corn waste at pH 3.2
3. Effect of pH on COD reduction by T. viride in corn 20
waste after 24 hours
4. Effect of temperature on COD reduction by I_. viride 22
digestion of corn waste after 24 hours
5. Reduction of carbohydrates, COD, nitrogen and 23
phosphate by T_. viride growing on corn waste
6. Effect of added nitrogen and phosphate on COD 25
reduction by T_. viride on corn waste
7- COD reduction in continuous culture fermenter of 28
corn waste by T. viride
8. Continuous digestion of corn waste by T. viride 30
9a. T. viride colony 35
9b. Penicillium colonies
9c. T_. viride and Penicillium
10. Disappearance of dissolved oxygen by T. viride 36
growing on corn waste in a continuous culture
11. Rat growth curves 39
12. Effect of pH on COD reduction by T. viride 185 in 42
soy whey after 24 hours
13. Effect of temperature on COD reduction by T. viride 44
185 in soy whey after 24 hours
14. COD reduction as a function of inoculum size 45
15. Rate of COD reduction as a function of inoculum 45
size
16. Fungal growth in soy whey predigested for 16 hours 49
with T. viride 185
vxi
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FIGURES
Page
17. Continuous COD reduction of HC1 soy whey by 52
G. deliquescens,
18. Inoculation-dilution technique for starting a 54
continuous digestion of soy whey by T^ viride
19. Continuous digestion of soy whey by G. deliquescens 56
20. Disappearance of dissolved oxygen by G. deliquescens 58
growing on soy whey in continuous culture
21. Activity of fungi on COD reduction of S02 soy whey 61
containing 415 mg SOg/l
22. COD reduction of SOg soy whey by A. oryzae 62
23. Effect of rapid passage of A. oryzae on COD 63
reduction of SOs containing soy whey at 710 mg
S03/l
24. Utilization of S02 by A. oryzae pregrown in the 65
presence and absence of SOs
25. Continuous digestion of S02 soy whey by 67
G, deliquescens
26. Continuous digestion of SOs soy whey by 69
G_. deliquescens
27. Continuous digestion of SOs soy whey by 71
G_. deliquescens
28. Weanling rat growth rates fed a standard casein 77
diet and a test G. deliquescens fungal diet
viii
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TABLES
Page
1. Name, Strain, and Source of Organism 9
2. Growth of Various Fungi on Corn Waste 17
3. Digestion of Corn Waste by Selected Fungi at pH 3.2 18
4. Chemical Composition of Corn Waste 24
5. Chemical Analyses of Corn Waste Before and After 26
Growth of T. viride 1-23
6. Reduction in the Chemical Components of Raw Corn 32
During Continuous Digestion by T. viride
7. Pigment Production Under Conditions of Nitrogen 33
and Phosphate Suboptimal for Growth of T. viride
8. Amino Acid Composition of Several Proteins 37
9. Digestion of Soy Waste by Various Species and Strains 41
of Fungi Imperfecti
10. Increase in Mass of T_. viride 1-185 Mycelium 43
Produced as a Function of Inoculum Size
11. Chemical Analyses of Soy Waste Before and After 46
48 Hours' Digestion by T. viride 1-185
12. Effect of Nitrogen Supplementation on Fungal 47
Digestion of Soy Whey
13. Residual COD as a Function of Dilution of Soy Whey 48
before Inoculation
14. Digestion of Spent Soy Supernatant by G_. deliquescens 50
15. Amino Acid Analysis of Two Fungal Strains Compared 59
to Several Standard Proteins
16. 24-Hour Reduction of COD and S02 in a Soy Whey 60
Containing 147 mg SOg/l
17. Growth of an A. oryzae Adapted to Sulfur Dioxide 64
at Varying Sulfur Dioxide Concentrations
IX
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TABLES
Page
18. Reduction of the Chemical Components of SOs Soy 72
Whey by G_. deliquescens
19. Rat Feeding Trial Casein vs. G. deliquescens Test Diet 75
20. Rat Feeding Trial. Fungal Mycelium as Sole Source 74
of Diet
21. Economy of Corn Waste Treatment 79
22. Economy of Soy Waste Treatment 80
x
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SECTION I
SUMMARY
Forty-five species of twelve genera of fungi were screened to select
those most capable of reducing the BOD of commercial corn and soy pro-
cessing wastes by converting the soluble and suspended organic matter
to mycelium. The screening led to the selection of rapidly growing
fungal strains that could reduce the BOD of the corn and soy wastes
from initial values of 4000 and 8000 mg/1, respectively, to <50 and
200 mg/1. The mycelium could be readily removed from the digested
waste effluents by a simple, coarse filtration. Trichoderma viride,
Gliocladium deliquescens, and either Aspergillus oryzae or G. deli-
quescens gave the best results on corn, soy, and SOa-containing soy
wheys, respectively.
The process proved adaptable to continuous fermentation and continuous
runs of many weeks duration were conducted. Sterile conditions were
not required and were used only in the first stage of inoculum transfer
to the liquid medium.
Maintenance of the fungal strain as the dominant organism seemed to be
dependent on the use of a relatively heavy inoculum, pH control in the
range of three to four (by addition of sulfuric acid), and feeding at
a high enough rate to prevent the culture from going into a stationary
phase with extensive sporulation and lysis. If these events occurred,
other organisms, including bacteria and, particularly, yeasts, appeared
in the fermentation in large numbers. It was usually possible to re-
establish the fungus if the period of starvation had not been too long
and if refeeding was undertaken judiciously. Recovery was achieved
-after a, .feed stoppage of up to eighteen hours, but longer interruptions
were likely to cause serious trouble. Loss of excessive mycelial mass
through dilution and washout occurred at very high feed rates.
Corn Waste
For corn waste, the optimal retention time, once the culture had
achieved heavy growth, appeared to be about twenty hours. Shorter re-
tention times were investigated to a limited degree, but washout of
fungus appeared to be occurring when the time was reduced to sixteen
hours.
These data were obtained at 19-24 C. The optimal temperature was about
30°C, but the temperature response curve was relatively flat; half the
maximum rate was achieved at either 10°C or 40°C.
Aeration requirements were modest, possibly because only a fraction of
the BOD was totally oxidized. The rest was incorporated into the my-
celium. About one pound of dissolved oxygen was required per seven
pounds of COD utilized. Agitation vigorous enough to keep the mycelium
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in a homogeneous suspension was required. If the mycelium clumped, it
became anaerobic in the center of the mass, and lysis occurred.
Inoculation of the continuous cultures was most smoothly accomplished by
adding a physiologically young culture to the fermentor containing one
part medium and eighteen to twenty parts water. As soon as the culture
was added, continuous feeding was begun at the desired rate. This pro-
cedure avoided large excesses of nutrients; this was desirable because
excess nutrients probably would have allowed competing organisms to be-
come established. Typically, an inoculum volume of about one-twentieth
the fermentor volume was used. The use of smaller amounts was investi-
gated only to a limited degree.
It was necessary to add nitrogen and phosphate to the corn waste because
of its low content of these nutrients. Some growth occurred without added
N and P, and smaller quantities than those used routinely might have sup-
ported adequate growth. Phosphate levels were regulated to control the
continuous fermentation of corn waste, but reductions in nitrogen sup-
plied were little explored because of the desirability of a high protein
content in the mycelium. The two levels of nitrogen most investigated
would have led to 45 and 90 percent protein in the mycelium if all had
been converted to protein. Nitrogen analyses of the mycelium produced
at the two levels of addition indicated 35 and 59 percent protein content,
respectively. At the lower level of addition, only negligible amounts of
nitrogen or phosphate escaped in the effluent. Excess phosphate was un-
desirable for several reasons. One is that phosphate is usually unaccept-
able in waste effluents; another is that a colored effluent was produced
at high phosphate -to-nitrogen ratios; and a third is that the fermenta-
tion was more stable and better controlled when the growth rate was
limited by phosphate supplies.
The combination of requirements for pH control and nitrogen addition con-
tributed to the total solids content of the effluent. If pH control on
the acid side had not been required, the nitrogen could have been added
as ammonia, contributing no residue. Part of the sulfate and sulfuric
acid additions was incorporated into the mycelium, but part appeared in
the effluent stream. This might cause difficulty in meeting stream
standards when highly concentrated wastes are treated. Similarly, the
low pH would be unacceptable in many instances.
Harvesting the mycelium was easy. The mycelium was recovered by gravity
filtration through a nylon mesh. When allowed to drip dry on the nylon
mesh, the mycelium contained only 80 percent water. Filtration by vacuum
was less successful because the mycelium became packed together and soon
restricted the water flow. Commercial vacuum filters in which the filter
cake is continuously discharged would probably work satisfactorily, but
were not tested.
The utility of the mycelium as a feed seemed promising in limited studies.
The amino acid composition was gratifying, particularly with regard to
high lysine, threonine, and tryptophan content. The amount of sulfur-
containing amino acids was lower than hoped for, but not too serious,
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since methionine fortification is within the realm of economic possi-
bility. The digestibility was excellent in weanling rats. Net nitro-
gen utilization was lower than ideal but was an artificially low figure
because all nitrogen was assumed to be in protein. Palatability to
rats was excellent. They avidly consumed even the pure fungal mycelium.
Yields of fungal mass were high, equivalent to about fifty percent of
the COD utilized.
Economic estimates are presently major extrapolations. The possibility
of at least breaking even (cost of processing the waste vs. return from
sale of product) seems reasonable.
Soy Waste
The reduction of BOD in soy wastes by more than 97 percent was accomp-
lished by the fungus strains used even in the presence of 700 ml/1 of
SOs. Fermentation reduced the S02 level by 96 percent. The residual
BOD seemed to be refractory to the fungal organisms. The mode of re-
moval of SOs was not investigated, but the sulfur did not appear as
increased sulfur-containing amino acids in the mycelial protein. The
BOD was further reduced by half in one set of continuous fermentation
experiments by adding a second stage fermentor containing mixed flora
obtained from a soil enrichment culture. Unlike corn waste, since no
nutrients needed to be added to the soy waste, no problems with added
inorganic ions remaining in the stream were encountered. Phosphate
and nitrogen were reduced by 70 and 90 percent, respectively, by
Gliocladium deliquescens, but remained higher than ideal.
Several of the operating parameters were similar to those for treating
corn waste. Sterile conditions were not required. The optimal pH was
between three and four, again achieved by the addition of sulfuric acid.
Optimal temperature was about 30°C, with half maximum rates at about
20° and 40°C.
Control of the fermentation to prevent appearance of BOD in the efflu-
ent or to prevent the culture from going into a stationary phase, thus
allowing the invasion of competing organisms, was more difficult than
for corn waste. This may have been because soy waste is a better
medium for competing organisms and because there was no need for addi-
tional nutrients which can be withheld to control growth, as with phos-
phate for corn waste. Stability was obtained by two expedients, both
aimed at maintaining an adequate balance between available nutrient and
mycelial mass. One expedient was to vary the feed rate in response to
variations in the COD level, which was never allowed to approach closer
than 200 mg/1 to the minimum attainable. The other was to remove my-
celium to maintain a constant amount in the fermentation. This was 3.2
to 3.7 g/1 when the COD of the feed was 10 g/1. A retention time of
about thirty hours was required.
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Aeration requirements were similar to those for corn waste: one pound of
dissolved oxygen per 5.5 pounds of COD utilized. The yield was about
fifty percent of the COD utilized.
The nutritional adequacy of the soy waste obviated the necessity of adding
nitrogen or phosphate.
Experience with harvesting the mycelium was the same as with corn waste.
The feed potentialities of the mycelium remain in doubt. The amino acid
composition was excellent, but the mycelium was unpalatable to rats.
Washing with alcohol made it palatable; with water did not. Systematic
feeding experiments were not conducted on alcohol-washed mycelium but
two rats consumed a diet of washed fungus, alone, for two days without
apparent deleterious effects. A brief feeding experiment was conducted
on mycelium grown on HC1 soy whey (no sulfur dioxide). The animals did
not grow as rapidly as the controls, but several explanations are pos-
sible. One is that the experiment was too short to permit recovery of
rate of gain after the first two days in which feed consumption was de-
pressed on the experimental diet. Another is that the digestibility of
the fungus was low. A third is a toxic factor. Toxic materials have
been reported in some Gliocladium strains. More experimentation is
called for. If the Gliocladium should prove unacceptable as a feed,
Trichoderma strains which showed growth on SOg-containing whey could be
reinvestigated. The Gliocladium strain was chosen because it grew faster
at high sulfur dioxide concentrations and rapidly reduced the COD.
The economics of the use of fungi on soy whey appeared more promising
than those of corn waste. One reason for this was that no additions of
nitrogen and phosphate were necessary. Another was the possibility of
year-round operation. The year-round operation does, however, add
another expense: heating will probably have to be supplied in northern
climates during the winter months. The cost of such heating depends on
the availability of waste heat from soy processing.
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SECTION II
INTRODUCTION
The Federal Water Quality Administration awarded Grant No. 12060 EHT
to North Star Research and Development Institute in August 1967 to
cover 70 percent of the funding of a study of "Us.e. of Fungi Imperfect!
in Waste Control". The remaining 30 percent of the funding was
provided jointly by the Central Soya Company, General Mills, Inc.,
the Green Giant Company, and the Ralston Purina Company. The study
was programmed for two years, ending August 31, 1969.
The objective of the research was to select rapidly growing strains
of fungi that would convert dissolved and suspended organic matter
in waste streams from corn and soy bean processing plants into a
mycelium that could be readily harvested by filtration. For the
process to be practical, it was necessary that the selected fungus
reduce the BOD value of the waste streams to a very low level and
that the mycelium have utility as a feed product. To accomplish
these objectives, it was necessary to select fungi which were
capable of establishing themselves as the predominant organisms in
nonsterile waste streams. Practical considerations required that
the organisms used be relatively insensitive to small variations
in temperature, pH, nutrients, and aeration. The mycelium would be
most valuable as a feed if it was of high protein content.
Economic considerations required that the organisms be established
with only minimum requirements for nutrient additions, pH adjustments,
aeration requirements, operational management, etc.
This report covers the study from its initiation on September 1,
1967, to its completion on August 31, 1969.
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SECTION III
BACKGROUND
For microbiological treatment of organic waste streams to be practical
it was necessary for the selected fungus to reduce the BOD to a low
level and for the mycelium to have utility as a feed or food product.
To accomplish these ends, fungi capable of establishing themselves as
the predominant organisms in nonsterile waste streams would be required.
They should also be relatively insensitive to small variations in tem-
perature, pH, nutrients, and aeration. The mycelium would be most val-
uable as a feed it if had a high protein content.
Several of the requirements for practical use of fungi in waste control
appeared from previous work to be met by species of fungi. Lilly and
Barnett, (22) , Cochrane, (23), Gray, et_ al. , (7,8), reported that many
of these fungi grew rapidly on sugar cane and sugar beet molasses as
well as on crude raw plant materials. Gray, (7,8), obtained fungal my-
celia containing 25 to 35 percent protein and with an amino acid compo-
sition comparable to casein. Limited tests showed some of the strains
to be nontoxic when fed to mice. Another reason for interest in Fungi
Imperfecti was the well known production of cellulolytic enzymes,
Mandels and Reese, (12,13). Many of the vegetable processing wastes
are known to contain cellulosic materials in suspension. Finally, the
demonstrated capability of these organisms to grow at pH values below
5, Lilly and Barnett, (22), and Cochrane, (23), offered some hope of
controlling competing organisms by conducting fermentations at low pH
values.
Waste- treatment at low pH values has been investigated very little.
Eckenfelder, (5), reported that most of the studies in the literature
concluded that biological waste treatment could not be conducted at low
pH and that waste degradation at pH values below 5 was not desirable.
There are, however, a few examples of low pH studies which, in the past,
showed excellent decomposition of waste. The growth studies of Pipes
and Jones, (17), using Geotrichum candidium and Sphaerotilus and the
studies of Cooke, e_t _al. , (3), showed reduction in organic matter and
BOD values as a result of increased fungal activity at pH values down
to 2.9. The studies of Cooke,' e± al., (3), were done with nine fungal
strains and showed the BOD reductions to be accompanied by significant
utilization of dissolved oxygen at the low pH. Brower and Gaddis, (2),
studied waste treatment by filamentous organisms at low pH values. Few
bacteria appeared at the lower pH values, but occasionally large numbers
of yeasts developed. The substrate used by these workers was a synthetic
composition of glucose, salts, and yeast extract. In the pH range of 6.5
to 7.0, they showed the presence of a large variety of organisms includ-
ing yeast, bacteria, fungi, and protozoa.
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An attractive reason for using fungi in an aerated continuous culture
system was that, in the conversion of carbohydrate from plant wastes to
mycelial protein, nitrogen and phosphate would be required for the trans-
formation. If plant wastes were low in these two materials, the final
effluent from the controlled waste conversion could be essentially free
of nitrogen and phosphate. These reductions would be added desirable
features to a system which degraded organic waste to a low BOD and
allowed for a harvestable protein.
Procedures of biological waste treatment are the oldest and largest ap-
plication of the so-called continuous fermentation. Certain troublesome
problems such as elimination of slowly decompo'sable substances cannot be
resolved without previous theoretical considerations on the dynamics &£
continuous processes. Even in modern texts on waste purification no men-
tion is made to the already extensive literature in this field. One of
the few workers who recognize the duality of waste treatment and continu-
ous processes is Herbert, (9), who included waste treatment in his dis-
cussions in continuous fermentations. According to Herbert, (9), all
systems of modern biological sewage treatment works are "open continuous
systems with feed-back".
One may even extend the scope of study of large continuous systems found
in municipal waste treatment plants and, hopefully, in the food processing
industry to even larger natural bodies of water where freely suspended
microbial cells are assumed to exist under near starving conditions. Here,
like sewage treatment plants, Jannasch, (10), points out that growth will
be limited primarily by low concentrations of suitable carbon and energy
sources. It is not inconceivable that continuous growth in natural waters
occurs at extremely low rates.
Earlier studies on growth rates in continuous culture [Novick, (15), and
Postgate and Hunter, (18)], showed definite minimal growth rates in con-
tinuous (chemostat) cultures. Thus, Novick, (15), grew a tryptophan-
requiring mutant of Escherichia coli in well supplemented media and found
the organism ceased to grow below a generation time of 15 hr at 37°C. He
assumed that unbalanced and discontinuous growth occurred at shorter re-
tention times in the chemostat, preventing establishment of a steady
state. Postgate and Hunter, (18), found steady states at far longer re-
tention times with cultures of Aerobacter aerogenes in studies of bac-
terial survival.
Jannasch, (10), reported indirect determinations of growth rates from
washout rates of bacterial populations in continuous culture. In this
manner, he hoped to estimate rates of microbial growth and transforma-
tions in natural waters. By using several organisms in the same chemo-
stat, he could evaluate the competition for the natural substrate as
well as for certain nutritional supplements.
Attempts will be made in these studies to understand the effect of reten-
tion time (dilution rate), washout rate and generation time (doubling of
fungal mass) of the continuous fungal culture system growing on industrial
waste. The objective will be to achieve a steady culture state where in-
flowing nutrient (waste) is reduced to the lowest possible organic level
and the mycelium is harvested as a feed product.
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SECTION IV
MATERIALS AND METHODS
Fungal Stocks
Fungal Stock cultures were maintained on Czapek-Dox, Sabouraud-dextrose,
sterile corn, and soy waste agar slants at 4°C. Stock cultures were
transferred every three months to freshly prepared agar slants. The
organisms used in these studies and their sources are listed in Table 1.
Table 1
Name, Strain, and Source of Organism
Fungus
Heterocephalum aurantiacum
Cladosporium unknown
Linderina pennispora
Dactylium dendroides
Paecilomyces elegans
Aspergillus oryzae
Gliocladium roseum
Gliocladium deliquescens
Morchella esculenta
Trichoderma viride
Tricho derma viride
f
Trichoderma viride
Myrothecium verrucaria
Streptomyces unknown
Coprinus unknown
Strain Number
1-9
1-75
1-100
1-108
1-134
1-14
1-30
1-31
1-23,1-184,1-185,1-186,
1-187,1-188,1-190,1-191,
1-192,1-193
M-114
QM-6a
QM-460
—
—
Source
W. Gray
W. Gray
W. Gray
W. Gray
W. Gray
W. Gray
W. Gray
W. Gray
W. Gray
W. Gray
Ralston-
Purina
Natick
Natick
*Soil
*Soil
Soil from Bushton, Kansas, soy bean fields.
The cultures marked Gray in Table 1 were selected by Dr. William Gray
from his stock culture collection in the Department of Botany, Southern
Illinois University. His selections were based on his past experiences
with these cultures in which he studied their ability to digest carbo-
hydrates from various sources (not corn and soy wastewaters).
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Before a fungal species was used for an experiment, it was transferred
from the agar slant stock culture to sterile neopeptone-dextrose broth
and incubated for 48 hours at room temperature. A broth culture of each
organism was also stored at 4°C for two or three weeks as a working pri-
mary culture before it was either discarded or transferred to fresh broth
for potential storage at 4°C in broth, the spores of the fungus were fre-
quently used as an inoculum into fresh neopeptone broth or into corn and
soy wastewaters. After one or two 24-hour transfers through corn or soy
waste media, the fungus was used as an inoculum for experimental purposes.
In several special cases, a secondary continuous digester was coupled to
a primary continuous soy digester described in a later section of these
methods. The inocula for the secondary digester were microorganisms con-
tained in a soil sample obtained from soy bean fields in Bushton, Kansas.
The culture medium was undigested residual organic effluent material re-
sulting from continuous fungal primary digestion of raw soy whey. Thus,
a continuous soil enrichment system was established to trap those orga-
nisms which were best able to grow on the primary effluent medium. This
soil enrichment technique resulted in the emergence, in the secondary
digester, of a streptotnycete and a basidiomycete which grew mutualistic-
ally to further degrade the primary effluent. Thus, the microorganisms
were selected naturally rather than from stock culture collections in
this special case. Both microorganisms were isolated in pure culture
and added to our stock culture collection.
Media
The media used in these studies were corn waste, HC1 (edible) soy whey,
and SOa (industrial) soy whey. These media were collected from plant
effluents in 5-gallon polyethylene containers, frozen quickly, and stored.
They were thawed immediately before use in a Heinicke Instruments Co.
dishwasher and, if used in part, the remainder was discarded.
Incubation
All batch-type cultures were incubated at room temperature (26° to 32°C)
on a New Brunswick double tier rotary shaker. Best growth results were
obtained on this shaker in Bellco flasks which had bottom indentations
to improve liquid mixing and, thus, aeration. Culture volumes ranged
from 100 ml to one liter per flask.
Otherwise, both batch and continuous cultures in corn and the soy wheys
were incubated at room temperature in laboratory fermentor apparatus
which was developed during the course of these studies.
-10-
-------
Continuous Culture Apparatus
A design of the apparatus is shown in Figure 1. One need only adjust
the inflow rate of waste'material (theoretically) to maintain the op-
erational efficiency of the system. The inflowing nutrient was ad-
justed to a rate which would maintain a constant fungal mass in a
steady metabolic state, where any increase in mycelium was balanced
by loss of mycelium at the product take-off point as shown in Figure
1. If optimal cultural conditions were maintained, one would expect
to feed the continuous system with waste medium at a rate which would
bring about a complete turnover of the wastewater volume. Hopefully,
the time required for a complete turnover of wastewater would be
shorter in a continuous culture than in a batch system because the
fungal culture would always be maintained in a physiologically young
state. In this state, metabolism of the waste nutrients would be at
a maximum continuous rate. In addition, mycelial harvest would also
be continuous.
The primary digestor was a polyethylene 25-liter carboy with the top
cut off and inverted into a 300 mm glass funnel. Holes were tapped
into the top (formerly bottom of the polyethylene carboy) of the di-
gestor for insertion of various probes and tubes. The bottom of the
funnel was either plugged or fitted with plastic plumbing parts for
admitting air, sampling the digestion mixture, or totally draining
the system. One stone sparger in a digestor of this configuration
was sufficient to provide any desired dissolved oxygen concentration
from 0.1 to 3.0 mg/1 and with enough agitation to maintain the cul-
ture in suspension.
In the diagram (Figure 1) of the digestor apparatus, squares enclosed
in dotted lines represent that part of the system which was contained
within a 4°C cold room. The raw feed was either pumped from the mag-
netically stirred feed tank to the digestor or elevated to allow
gravity feeding at timed intervals controlled by a solenoid timer
switch on the feed line. If more sensitive feeding controls were
required, selected lengths of tygon tubing were inserted into the
feed line to increase flow resistance. Since this tubing length con-
trol frequently plugged when very low feed rates were used, it was
discarded in favor of the solenoid timer switch mechanism. These
simple solenoid timers were constructed in our shop and were superior
to any of the pumps or other devices that were tried at various times
during the course of these studies.
Since some foaming was usually present during the early stages of waste
digestion and before a low (near starvation state) COD was established,
the entire digestion system was closed and air, from the sparger,
served to gently push the fungal contents out the effluent tube. The
air, which produced a slight positive pressure in the digestor, escaped
through the nylon filter shown in Figure 1. The nylon filter served
to entrap the fungal mycelium and, as the mycelial mass increased,
developed efficient dewatering features. Either the effluent liquor
-11-
-------
Pressure gage
Rotaraeter
Feed
Tpump Sampling
_Jk
Raw fc
c-
!«
z
u— '
id
^>
._—L—-
port
^-Stirrer
Primary
continuous
digester
Nylon
liter
Primary effluent
•^t—•
Secondary
continuous
digester
Fig. 1. Fermentor apparatus for continuous digestion of corn and soy waste by fungi.
-------
passed through the nylon filter to the laboratory drain or the liquor
was further processed (in the case of soy whey) through a secondary
digester. This secondary digester consisted of a three-neck, 4-liter,
round-bottom distilling flask fitted with rubber stoppers and the re-
quired feed, air, and effluent lines.
When changes in the nutrient composition or pH were desired during
continuous digestion of the industrial wastes, such changes were made
via additions to the feed reservoir contained at 4°C.
Analytical Measurements
Cultural
Measured liquid samples were removed from the continuous digester sys-
tem and vacuum filtered through tared No. 4 Whatman filter paper in a
Buchner funnel. The filter papers containing the mycelium were dried
to constant weight at 90°C. Fungal mycelial samples collected by fil-
tration in this manner were recorded as mg dry weight/1 of digester
liquor.
Microscopic examination of the effluent corn samples did not reveal any
corn particulates which would otherwise be trapped on the filter along
with the fungal mycelium. The COD of the filtrate was not increased
when a small effluent sample (50 ml) from the continuous corn digestion
system was filtered and washed through a nylon stocking instead of a
Whatman No. 4 filter paper. We believe, therefore, that at least in
the case of corn digestion the effluent COD measured after filtration
was not reduced by physical trapping undigested corn particulates in
the fungal matte.
The COD of soy filtered effluents may have been reduced by a small per-
centage due to trapping soy particulates on the Whatman No. 4 filter
paper. Microscopically, occasional undigested soy particulates, large
enough1to be trapped with the fungal mycelium during filtration, were
observed. When these effluent samples (50 ml) were filtered and washed
through a nylon stocking rather than Whatman No. 4 paper, approximately
10-15 percent increase in COD was repeatedly observed.
Physical
Aliquots of filtrates from these liquid culture samples were dried at
90°C in tared aluminum pans to constant weight for determination of
total solids. When ash determinations were desired, other aliquots of
these filtrates were dried at 90°C to constant weight in tared nickel
crucibles and then heated at 600°C for 4 hours.
Chemical
Total phosphates in corn and soy wastes were determined by the method
of Fiske and Subbarow,(6).
-13-
-------
The method of Lowry, e_t aJL. , (11), was used for protein determination.
Human serum albumin was used as a protein standard. Proteins were sep-
arated from other nitrogenous compounds by trichloroacetic acid precip-
itation.
Total carbohydrates were determined by the phenol sulfuric acid method
of DuBois, et al. , (4), and glucose by the Nelson, (14), modification of
the Somegyi method. Standards were starch and glucose. Other carbohy-
drate analyses such as the anthrone and cysteine-sulfuric acid methods
were found to be unsuitable in the presence of ammonium salts.
Chemical Oxygen Demand (COD) was determined by the methods described in
Standard Methods for the Examination of Water and Wastewater, (19). The
COD is defined as the oxygen consumed by organic constituents in a water
sample in an oxidation reaction with a strong oxidizing agent, i.e.,
chromic acid or bichromate-sulfuric acid at boiling temperature.
Biochemical Oxygen Demand (BOD) procedure was carried out as described
in Standard Methods for the Examination of Water and Wastewater, (21) .
BOD is defined as the biochemical oxygen demand in five days, i.e., the
oxygen consumed by the respiration of the microorganisms in a water
sample within five days at 20°C. The seed samples used in the BOD deter
minations were obtained from a municipal sewage source in Montgomery,
Minnesota, from Mississippi mud mixed with fertile garden soil, from
Minnesota river mud in an area of the river where corn wastewater was
discharged, and from Ohio river mud in the area of Louisville, Kentucky,
where soy wastewater was discharged.
determinations were made according to the standard procedure described
in Official Methods of Analysis of the Association of Official Agricul-
tural Chemists, (16). Total nitrogen determinations were made by the
micro-Kjeldahl procedure. Nonprotein nitrogen (NPN) was also determined
after precipitating the protein.
Amino acid analyses of the dried fungal mycelium were performed with the
Beckman Amino Acid Analyzer after hydrolysis of the fungal samples. These
analyses were performed by the Central Soya Chemurgy Research Laboratories,
Sulfate, chloride, and nitrate analyses were conducted according to
Standard Methods for the Examination of Water and Wastewater, 1956.
Feeding Studies
Weanling rat feeding studies were carried out primarily to determine
whether or not toxic manifestations were inherent in the fungal proteins.
Other considerations were digestibility and utilization of the fungal
protein. A standard weanling rat diet was supplied by Nutritional Bio-
chemical Corp. (NBC) and consisted of 23 percent casein, 2 percent alpha-
eel, 59 percent starch, 10 percent vegetable oil, 4 percent salt mixture ,
2 percent vitamin mixture and 0.1 percent methionine. A second NBC diet
-14-
-------
formulated for these studies contained 46 percent dried fungi (T\
viride contained 50 percent protein therefore this diet, after mixing
contained 23 percent protein), 44 percent starch, 2 percent vegetable
oil, 3 percent salt mixture, 2 percent vitamin mixture and 3 percent
L-amino acids. The additional amino acids supplemented the fungal
protein to give it the same amino acid composition as casein.
Analyses of total carbohydrate, ash, and lipid content of the fungus
are shown in the test. In adjusting the composition of the diet-con-
taining fungus to make is as nearly equivalent as possible to the con-
trol diet; the fat, lipid, and carbohydrate content of the fungus were
taken into account.
The difference between the standard and test diet was probably large
in the area of vitamin content. Analyses for vitamins in the fungus
were not carried out, except for niacin.
The weanling rat feeding experiments were carried out in the following
manner: three rats were placed on the standard diet and three were fed
the test (fungal) diet. Each rat was placed in a separate metabolic
cage. Fecal material was collected by means of a tube container at-
tached to the rat's tail. Urine was collected free of fecal contamina-
tion. Feed weights and rat weights as well as total fecal and urine
excretions were measured or collected daily. All six rats were started
on the standard casein diet containing 1 percent chromic oxide. After
one day, three rats were fed the standard casein diet without chromic
oxide, and the three test rats were fed the fungal diet. Fecal collec-
tion was begun when no more chromic oxide appeared to color the fecal
pellets. This loss of green stain in the feces occurred after approx-
imately 24 hours. Thus, only fecal material from the rats eating the
experimental diets were collected, and these feces were free of dietary
material eaten prior to this experiment. Nitrogen analyses were carried
out on selected urine and fecal samples as well as on the dietary mate-
rial taken daily during the course of the feeding trial. In addition
to these analyses, body weight gains and daily physical examinations
for ruffled fur, scaly feet, encrusted eyes and nose, retarded incisors,
etc. were taken. These measurements supplied the information required
to evaluate the fungal diet as to its digestibility, protein efficiency,
and toxicity.
-15-
-------
SECTION V
RESULTS
Corn Waste
Fungal Strain Selection
Selection of the most effective organisms for use on liquid corn waste
was conducted initially without adjustment of pH of addition of nutri-
ents. Screening for rapid growth was carried out in 30 ml volumes of
corn waste placed in 125 ml Erlenmeyer flasks and incubated at 27°C on
a New Brunswick rotary shaker at 140 oscillations/min. Results such
as those shown in Table 2 were obtained. All fungi showed an initial
lag before growth, and competition with the natural bacterial and yeast
biota initially proved difficult. This is illustrated in Table 2 by the
differences between mycelium production in the sterile and nonsterile
corn waste medium.
Table 2
Growth of Various Fungi on Corn Waste
pH 7.2
Fungus
Cladosporium
Linderina pennispora
Dactylium dendroides
Paecilomyces elegans
Aspergillus oryzae
Tricho derma viride
Gliocladium roseum
Gliocladium deliquescens
Fungal Mass After 6 Days
Sterile
(mg/30 ml)
15.2
17.0
15.1
21.0
23.8
18.3
10.1
16.1
Nonsterile
(mg/30 ml)
8.4
5.5
8.0
5.6
4.4
11.0
10.0
10.0
Adjustment of the pH to lower values permitted a more successful compe-
tition with the natural corn waste biota. The pH of the corn waste as
received from Green Giant was 7.2. When this pH was reduced to 3.2
with HsSO.4 before inoculation, rapid growth and COD digestion occurred.
The reduction in COD and total carbohydrate at pH 3.2 by T. viride is
shown in Table 3.
-17-
-------
Table 3
Digestion of Corn Waste by Selected Fungi at pH 3.2
Organism
Raw waste
Natural biota
Trichoderma viride
Gliocladium deliquescens
Paecilomyces elegans
COD
mg/1
0 hr
2168
—
—
40 hr
—
1932
482
546
209
Carbohydrates
mg/1
0 hr
1420
—
—
40 hr
—
800
320
440
70
The fungal strains were next subjected to serial transfers through non-
sterile corn waste to achieve even more growth and COD reductions. The
effectiveness of the serial transfers in flask cultures is shown in
Figure 2. A considerable increase in COD reduction was achieved during
the second transfer. A fourth transfer (not shown) gave no further re-
duction in COD. Similarly increased rates in reduction also occurred
for carbohydrate, protein and phosphate. The results obtained by grow-
ing these organisms through a series of rapid transfers on nonsterile
corn wastes showed that COD reduction, carbohydrate reduction, phosphate
reduction, and fungal mass were successfully increased by this transfer
selection procedure.
pH Effects
It was observed during these transfer fla-sk experiments that lowering
the pH provided better fungal growth. Bacteria and yeast were depressed
at the lower pH levels. A systematic study of pH effects on COD reduc-
tion by T_. viride was made in shake flask cultures. Adjustments of pH
were made by addition of 1 N HC1 or 1 N NaOH. A heavy inoculum was used
(1.5 mg/ml). The effects of pH on COD reduction after 24 hours are shown
in Figure 3. The optimal pH lies between 3 and 4. This agreed well with
results from other laboratories where T. viride was studied in a variety
of media and with the finding that the optimal pH was between 3 and 3.5.
In view of projected pilot plant studies with continuous fermentation of
nonsterile corn waste where a required initial holding period would
likely occur before fungal digestion took place, we undertook an exami-
nation of pH changes that might be expected during this corn waste hold-
ing time. Several 5-gallon polyethylene containers of corn waste were
allowed to remain at room temperature for 8 to 48 hours. The pH of
these 5-gallon samples dropped from 6.6 to 4.5 after 12 hours, and then
the pH slowly climbed to 7.0 by 48 hours. Therefore, continuous fermen-
tation of corn waste from a holding reservoir where the pH initially
dropped to a level of 4.5 to 5.0 may provide optimal pH conditions during
-18-
-------
2000
1500
§ 1000
o
500
0
Natural Biota | T
>- _ Q^ o
^ \ \
anaerobic j^ *
aerobic
1
0 25 50
0
. viride
1
\°
3 *fi
1
25
1-23 |C.
1
O
>
\
\\
b
|
50
0
deliquescens
t
\
\>°
\\
3^
1
25
1-3^ P.elegans
V
50 25
0
1-134
\
5(
Hours Incubation
Fig. 2. Strain selection of fungi for rapid COD reduction on corn
waste at pH 3.2. Numbers on lines refer to the number of fungal transfers.
-19-
-------
100
90
80
70
C!
O
•H
1 60
T3
•s
§ 50
5 40
ti
s
6 3
fc •*
10
io
o
4
e
Fig. 3. Effect of pH on GOD rtductlan by
T. viride in corn wast* after 24 hour*.
-20-
-------
fermentation. In several shake flask experiments it was shown that use
of corn waste that had been allowed to stand in a static state for 12
hours before use as a culture medium, dropped to pH 3.5 during the first
24 hours of fungal fermentation. This was the period of time where
fungal growth and COD digestion occurred at the most rapid rates. Ad-
justment of the waste to pH 5.1 with mineral acid before use had a sim-
ilar effect.
Temperature Effects
The effective temperature range for active digestion of the corn waste
is shown in Figure 4. Temperatures between 18° and 35°C resulted in
ninety or more percent reduction of COD. This would indicate than a
continuous fermentation would do well under outdoor conditions in the
late spring, summer, and early fall. However, below 18° or above 35°C
the percentage COD reduction decreased rapidly. The pH was held at
3.2 and other experimental conditions were the same as described for
the pH experiment, except that temperature variation replaced pH vari-
ation.
Nutrient Additions
It was observed in certain shake flask experiments that the COD, carbo-
hydrate, Kjeldahl nitrogen, and phosphate were not reduced very rapidly
after 40 hours incubation. These experiments indicated that either a
toxic by-product of the fermentation was produced or a deficiency in a
required growth nutrient was preventing the COD and carbohydrate diges-
tion from going to completion. The results of these studies (Figure 5)
showed that near exhaustion of at least two essential nutrients (phos-
phate and nitrogen) occurred after 40 hours. At a time when appreciable
amounts of COD (22 percent) and carbohydrate (28 percent) remained, low,
perhaps metabolically limiting, levels of phosphate and nitrogen were
detected. Thus, the possibility of a toxic by-product appeared to be a
less satisfactory explanation for the residual COD after 40 hours of
fermentation than the exhaustion of required growth nutrients.
To obtain additional information on the nutritional composition of the
corn waste growth medium, certain chemical analyses were performed. It
was hoped that more knowledge of the chemical composition of this waste
would aid the design of future experiments to study and lead to further
reduction of the residual COD. Also, knowledge of the chemical composi-
tion would act as a basis for determining what nutrient additions, other
than nitrogen and phosphate, might be required to effect complete metab-
olism of the corn waste. The results of the chemical analyses of corn
waste are shown in Table 4.
-21-
-------
ipo -
90
80
8 70
H
O
I 60
§
H 50
H
1 40
w
PM
30
20
10
0
i r
10 15 20 25 30 35 40 45
TEMPERATURE (°C)
50
Fig. 4. Effect of temperature on COD reduction by
!_._ viride digestion of corn waste after 24 hours.
-22-
-------
100
O
H
H
s
8
(^
w
10
20 30
HOURS AT pH 3.2
60
Fig. 5. Reduction of carbohydrates, COD, nitrogen and phosphate by
vtride growing on corn waste. Rates of reduction of these materials were
markedly reduced after 40 hours.
-23-
-------
Table 4
Chemical Composition of Corn Waste
Chemical Constituent mg/1
COD 2030
BOD5 1640
Protein 50
Carbohydrate 1360
Nitrogen (Kjeldahl) 48
Nonprotein nitrogen 38
Chlorides 784
Nitrates 0.6
Sulfates 120
Total phosphate 31
Total soluble phosphate 30
Orthophosphate 22
Total solids 3560
If one can equate BOD to chemical weight on a quantitative basis, it would
appear that 83 percent of the BOD is contained in the carbohydrate faction.
Both protein and total nitrogen are low. The effectiveness of nitrogen
and phosphate additions in increasing fermentation rates was therefore
explored. When T. viride 1-23 was grown on corn waste and nitrogen was
added as ammonium sulfate to cultures adjusted to pH 4.5, the results
shown in Figure 6 were obtained. The COD's after 24 hours were in the
900 to 1100 mg/1 range. They were further reduced by longer fermentation
time to less than 200 mg/1. Thus, a three-fold increase in nitrogen con-
centration (as ammonium sulfate) reduced the residual COD (Figure 6) ap-
proximately 15 percent more than was obtained before its addition.
Phosphate concentrations also appeared to be low, and phosphate additions
were studied. It was observed that phosphate as NaH2P04, alone, did not
increase the total COD reduction significantly. In combination with ni-
trogen, however, phosphate reduced the COD at a somewhat more rapid rate.
This is also shown in Figure 6. The ratio of N to P most effective in
reducing COD was 90 u.g (NEi)2S04/ml and 5 ng NaH2P04/ml or approximately
a 20:1 ratio for these two salts. Further increases in nitrogen and
phosphate concentrations failed to increase the digestion of the residual
COD.
These experiments showed that at least two required metabolites which
had limited COD digestion were deficient in the original corn waste medium.
In all these shake flask experiments, the pH had to be adjusted by the
operator from time to time. This pH adjustment of 3.2 to 3.5 was re-
quired after 8 hours in the flasks containing no additions, and after
36 hours in the flasks containing additional nitrogen. One pH adjust-
ment was made after 48 hours in flasks containing nitrogen and phosphate.
After 60 hours, the COD in all the flasks began to increase, and a micro-
scopic examination of the fungal contents revealed considerable lysis and
sporulation. -24-
-------
2500
2250
2000
1750
1500
E 1250
o
o
o
1000
750
500
400
300
200
100
0
None
O Natural biota
• T. virlde
90
5 Hg NaHaP04
10
20
30
HOURS
40
50
60
Fig. 6. Effect of added nitrogen and phosphate on COD reduction by
viride on corn waste. Concentrations of nutrients are indicated in
of culture. Nitrogen and phosphate additions are as salts
and NaHP0 in the concentrations shown above.
-25-
-------
Residual COD
Since a small residual COD remained even after prolonged digestion fol-
lowing the addition of nitrogen and phosphate, chemical analyses were
conducted to try to determine the nature of the unutilized residue.
The chemically analyzed material came from a 48-hour batch fermentation
with T. viride. The pH of the culture was maintained at 3.5, and 90 p,g
of (NH4)2S04 and 5 \ig of NaH2P04 were added per ml. The inoculum con-
tained 0.5^ mg/ml (wet weight) of T_. viride 1-23. These growth conditions
were those found best for digestion of corn waste liquors in the previous
experiments. Table 5 shows that the nitrogen was depleted and the phos-
phate reduced to low levels.
Table 5
Chemical Analyses of Corn Waste*
Before and After Growth of T. viride 1-23
Batch (Shake) Culture
Analytical Procedure
COD
BOD
Nitrogen - Kjeldahl
NPN
Protein
Carbohydrate
Chlorides
Sulfates
Nitrates
Nitrites
Total phosphate
Total soluble phosphate
Ortho phosphate
Total solids
Ash
Before
Fungal
Treatment
mg/1
2030
1640
48
38
50
1360
784
12P
0.6
0.008
31
29
22
3560
660
Fungal
.,,... Treatment
Additions , 0 ,
48 hrs
mg/1 mg/1
210
100
19 0.8
0.6
2
70
240
260 80
—
—
5
4 3
0
405
233
Analyses after 48 hrs were performed on samples which were filtered
through a single layer of Whatman No. 4 filter paper.
These chemical analyses suggest that further reduction in the unutilized
COD would require additional nitrogen arid phosphate. The remaining car-
bohydrate constituted 70 percent of the remaining BOD and 35 percent of
the COD. In previous experiments, additional nitrogen and phosphate, be-
yond that shown here, did not result in further COD reduction. Therefore,
either the remaining carbohydrate was refractile to digestion by this
fungus or metabolites, in addition to nitrogen and phosphate, were required,
-26-
-------
In one experiment to resolve this problem, fresh boiled yeast extract
and mineral salts in the form of an ashed suspension of fungus mycelium
were added to raw corn waste in flasks containing additional nitrogen
and phosphate. The flasks'were inoculated with 0.5 mg/1 on a dry weight
basis of T. viride mycelium, and the pH was adjusted to 3.5. Although
the COD was reduced only slightly (180 mg/1) after 48 hours, the BOD
dropped to 50 mg/1, and the carbohydrate was 30 mg/1. It appeared,
from these findings, that a near complete digestion of the raw corn
waste by T_. viride could be achieved if an extensive study was made to
find other exhausted metabolites which were growth limiting. This ex-
periment showed that some or all of these exhausted materials were pre-
sent in the yeast extract and fungal ash. What would also be required
would be a quantative nutritional balance of these growth-limiting-
materials before complete digestion of all the BOD was realized.
Continuous Fermentation
A continuous culture apparatus was developed in our laboratories to study
the conditions required to maintain continuous corn waste digestion by
J. viride 1-23. A diagram of this apparatus appeared as Figure 1, page
12.
The continuous culture was operated to obtain additional mycelium for
feeding trials and to obtain additional information on the performance
and stability of T_. viride digestion on raw corn waste under continuous
conditions. Initially, the system was started with 18 1 of raw corn
waste and a T\ viride inoculum of 0.01 g of mycelium/1 at a pH of 3.2.
Continuous feeding was begun after 40 hours, by which time the initial
COD had been reduced from 3976 mg/1 to- 1260 mg/1. Feeding was set at a
rate of 7.5 ml/min. in the 18-liter'fermentation vessel (average reten-
tion time of 40 hours). Aeration was conducted with two stone spargers,
with additional mixing by an air lift. Nitrogen was added to the feed
as ammonium sulfate at a level of 1.0 gm (NH4)2S04/i and sodium dihydrogen
phosphate was added at a level of 0.5 gm NaH2P04/l; 0.1 ml of sulfuric
acid/1 was added to the feed for pH control. This amount of acid re-
duced the pH of the raw corn waste from 7.1 to 5.2. Additional reduc-
tion of the pH was accomplished by the continuous fermentation itself,
and the pH remained fairly constant between 3.2 to 3.5. The results of
the COD reduction in this experiment are shown in Figure 7.
This initial experiment was in operation for ten days. Competition from
bacteria and other fungi was minimal. The initial number of bacteria
was., approximately 105/ml and, after eight days, was reduced to <103/ml.
We believed this was due to utilization of the organic nutrients by the
T_. viride and to the presence of protozoa (chiefly Paramecia) which
rapidly removed the bacteria. The experiment was concluded when the
COD began to increase slowly from 650 mg/1 at the fifth day to 800 mg/1
at the eighth day and then rise to 1500 mg/1 during the ninth and tenth
days. Microscopically it was observed at the eighth day that the my-
celium had begun to sporulate and fungal lysis was taking place. In
addition, pink pigmentation appeared at the fifth day and intensified
until the fermentor was reddish purple on the eighth day of operation.
-27-
-------
o
8
00
I
4000
3500
3000
2500
2000
1500
1000
500
-o—
1
1
O Continuous
C
345
taqrt (14-Hour F»rio*t)
8
Fit* 7* COD reduction in continuous culture fonMnter of corn w««t« by 1± virid*.
-------
As seen in Figure 7, the COD was initially reduced 60 percent over the
first 18-hour period. The solid line representing Area A in Figure 7
shows this initial digestion. The system operated initially as a batch
fermentor and the dry weight of fungal mycelium in the 18 liters in-
creased from 0.18 mg/ml at zero time to 0.66 mg/ml at the first sampling
point.
The total volume of corn waste treated during the 8-day period was 65
liters, and 59.15 grams of fungal mycelia were recovered. Areas B and
C in Figure 7 represent feed rate adjustments to 20 ml/min. at B and to
14 ml/min. at C. This latter figure represents an 18-liter volume turn-
over every 21 hours. Areas D and E were'intermittent batch and continu-
ous feed operations brought about by plugged feed lines and occasional
stoppages in the final effluent line. During this period the mycelial
dry weight increased from 0.66 mg/ml to 1.5 mg/ml. Areas F and G
(Figure 7) were also troublesome operationally and various feed pumps,
feed rates, collection devices, and samplers were tested. The aeration
device was plugged by the fungal mass during the first 8 hours of oper-
ation. Aeration was erratic from that time on. Stone spargers were
used to replace the aeration system.
Several results summarize this first attempt at a continuous culture
system and bear further explanation: (1) Stability of the pH at 3.2
to 3.5 without acid addition was found during the periods when the sys-
tem operated continuously. (2) The bacterial flora of the system were
approximately 10s/ml initially, <104/ml in area D of Figure 7, and
<103/ml at the end of the experiment. No yeast cells were observed at
any time during the 8-day run. (3) The protein composition of the fun-
gal samples taken at areas D, E, and F ranged from 40 to 45 percent on
a dry weight basis. These protein results were carried out by the Folin
and micro-Kjeldahl nitrogen methods, multiplied by 6.25 after subtraction
of the nonprotein nitrogen.
More extensive studies on the continuous digestion of raw corn waste
were conducted over a 140-day period. The fermentor was the same used
previously but with numerous mechanical modifications: pumps, aeration
devices, switches, etc.. Special attention was given to the nitrogen
and phosphate concentrations during this continuous experiment in view
of previous problems with ecological shifts in the fungal population
resulting in red pigment production. Also, the difficulty of maintain-
ing a near steady state culture was controlled by the level of phosphate
addition.
Behavior of the fermentation is illustrated in Figure 8 where points A,
B, C, and D refer to unstable conditions which developed during the
course of the fermentation. A and B were a result of feedline blocks
resulting in fungal starvation. C and D indicated times when the feed
pump stuck in the open position, causing a rapid flow of raw corn waste
and thus a washing-out of the fungus. When the pump errors were cor-
rected, a period of time was required before the fungus increased in
mass and again reduced the COD. Increased COD at the peaks between D
and E were a result of passage into the stationary phase, fungal lysis,
-29-
-------
r
3.5
3.0
— 2.5|
O
x
2-0
1.5
1.0
0.5
-Batch
D
I I I
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140
DAYS
Fig. 8. Continuous digestion of corn waste by I. viride.
-30-
-------
and related fungal cytoplasm. At point E (starting.at the 120th day),
the digestion was under the control of a limiting nutrient - phosphate.
From the 120th to 140th day, the continuous culture was strikingly stable,
as shown in Figure 8. The (NHOaSOi* added to the 18-liter digestion was
10 g or 0.55 g/1 or approximately 0.12 g N/l. Control of the fungal di-
gestion was accomplished by maintaining the phosphate concentration in
the digestion tank close to zero. This was done by assaying the phos-
phate concentration in the digester and adjusting the phosphate addition
to the feed. It was found that a concentration of 25 mg/1 of NaE^PCK or
approximately 20 mg phosphate/1 added to the feed reservoir resulted in less
than 1.0 mg phosphate/1 in the final digester effluent. Limitation of the
concentration of this required nutrient resulted in a stable fungal culture
in the digestor. This stability effect produced by phosphate is shown in
the continuous digestor system during the last days of the plotted data
(E) of figure 8. At this time the fungal mass was 1.9 to 2.1 g/1, the
residual solids 1.2 to 1.3 g/1, and the COD 150-180 mg/;. The fungus
appeared mature with homogeneous cytoplasm, budding at the hyphal tips,
and a white-tan color in the digestor, while maintaining a constant pH
of 3.1 to 3.3. The bacterial contamination, not visible microscopically,
was reduced from 8.5 x lOVml in the feed tank to 3.1 x 103/ml in the
digestor. No yeasts or protozoa were observed. The feed rate averaged
17 ml/min (ranging between 15-20 ml/min.), which corresponds to a turnover
time of 18 to 20 hours.
More than five pounds of dry lyophilized T_. viride fungus was collected
during the course of this continuous corn digestion and was mixed, as
the protein source, with a prefemulated rat feed which lacked only a
source of protein. The use of this fungal material as a feed for wean-
ling rats will be discussed later.
Chemical Composition of the Effluent
During the last 3 weeks of the continuous digestion (Figure 8) a chemical
analysis was performed on the effluent. The results of these analyses
are shown in Table 6. Although the original raw corn waste (Figure 8)
had a COD of 3750 mg/1 at the beginning of the continuous operation,
later feed materials from the corn processing plant were more concen-
trated, and the COD had increased to 5200 mg/1. Both nitrogen and
phosphate were reduced to very low values, by fungal growth, as was the
BOD.
-31-
-------
Table 6
Reduction in the Chemical Components of Raw Corn
During Continuous Digestion by T_. viride/
Test
COD
BOD5
*CHO
Protein
Nitrogen (Kjeldahl)
Phosphate (Total)
Sulfate
Fungi
Solids
Ash
Raw
mg/1
5200
3976
3500
200
96
32
120
0
4000
980
Addition
mg/1
116
20
280
Effluent from
Continuous
Treatment
mg/1
195
35
64
7.5
2.4
<1
210
2200
760
510
Percentage
Reduction
96.2
99.2
98.2
96.0
98.8
>98.0
48.0
81
52
CHO = total carbohydrate as determined by the phenol-sulfuric acid method.
'Analyses performed on samples of the effluent were made after filtering
through a single layer of Whatman No. 4 filter paper.
Pigment Production
During the course of some of these studies where nitrogen and phosphate
additions were made to continuous cultures of raw corn waste, a pink to
red pigment occasionally developed. This usually took place after three
to five days incubation. Since it would not be desirable to have red ef-
fluents present in scaled-up waste digestion systems and because the pig-
ment may possibly contribute an undesirable factor in fungal feeding ex-
periments, several investigations were made to learn something of the
nature of this pigment production.
A series of shake flasks containing raw corn waste inoculated with T_.
viride were grown as in previous experiments. Specified additions of
nitrogen and phosphate were made to the appropriate flasks, as shown in
Table 7. Incubation was continued for 48 hours at 26°C at two selected
pH's. Table 7 shows that both pH and phosphate were involved in produc-
tion of the red pigment. In instances (Table 7) where pigment was formed
at pH 2.5 and not at 3.5, one could drop the pH from 3.5 to 2.5 and ob-
serve the pigment. Conversely, the pigment intensity could be lessened
by raising the pH from 2.5 to 3.5. In flasks where no pigment produc-
tion was observed, prolonged incubation at pH 2.5 did not result in pig-
ment. It thus appeared that less than optimal growth conditions in the
presence of excess phosphate resulted in pigment formation. When opti-
mal growth conditions for I_. viride were established, as in the cases
where higher levels of nitrogen were used with the phosphate - no pig-
ment production occurred.
-32-
-------
These results led us to examine the fungal contents of the flasks with
the thought that, since the T. viride growth system was not a pure cul-
ture, there may have been other pigment producing organisms which were
favored under certain growth conditions which were suboptimal for T_.
viride. (NH4)2S04 and NaH2P04 were added in concentrations to supply
the nitrogen and phosphate shown in Table 7.
Table 7
Pigment Production tinder Conditions of Nitrogen and
Phosphate Suboptimal for Growth of T. viride
(NH4)2S04
p,g N/ml
0
0
0
0
0
0
40
40
40
40
80
80
120
120
160
160
300
300
NaH2P04
|ig P04/ml
0
0
9
9
18
18
0
0
18
18
18
18
18
18
18
18
18
18
PH
2.5
3.5
2.5
3.5
2.5
3.5
2.5
3.5
2.5
3.5
2.5
3.5
2.5
3.5
2.5
3.5
2.5
3.5
Pigment
none
none
pink
none
pink
pink
none
none
red
red
pink
pink
pink
none
pink
none
none
none
Fungus
mg/ml
0.81
0.78
0.84
0.88-
0.97
0.90
1.02
1.00
1.11
1.13
1.21
1.25
1.26
1.20
1.35
1.46
2.28
2.65
COD
mg/1
650
700
670
690
680
700
710
605
370
295
315
285
200
180
220
208
185
140
Microscopic examination of the fungal organisms and streak plating on
Sabouraud dextrose agar plates were done from all experimental flasks
used to collect the data shown in Table 7. The flasks with pigment ap-
peared to contain a mixed population of fungi when viewed microscopic-
ally. This was not the case with flasks which showed no pigment at
either pH 2.5 or 3.5. In addition, a large proportion of T_. viride
mycelium was undergoing sporulation in the pigment producing flasks.
The streak plates from pigmented flask cultures contained a mixture of
-33-
-------
T. viride colonies and green colonies of another fungus. Also, much red
pigment was observed on these plates. Streak plates from flasks which
showed no pigment production contained only an occasional green colony
among the numerous T. viride colonies. The fungi were isolated in pure
culture and cross streaked on additional agar plates. The results are
shown graphically in Figures 9a, 9b, 9c.
These plating experiments showed that the pigment was produced as a re-
sult of interaction between a fungus occurring naturally in the corn waste
and T_. viride. In cross streaking experiments such as those illustrated
in Figures 9a, 9b, the pigment occurred only at the junction of the two
cultures and was produced by the contaminating fungus. This fungus grew
on Sabouraud dextrose agar plates as round colonies with fluffy white
borders and a deep yellow zone inside the border with a dark green center.
Microscopically, we observed conidiophoral branches terminated by metulae
at the tips of which were clusters of phialides producing spores in par-
allel chains. This fungus was thus a species of Penicillium.
These studies also illustrated the ecological checks and balances which
exist in such a mixed population. Understanding the factors controlling
the mixed flora is necessary to controlling the fermentation.
Studies of pigment production leading to its elimination from the contin-
uous fermentation were interesting in that optimal T. viride growth con-
ditions of pH, nitrogen, and phosphate were conditions which apparently
inhibited growth of the contaminant fungus and thus precluded pigment
formation.
Dissolved Oxygen Utilization
During the early period of the continuous fermentation of raw corn waste,
the dissolved oxygen supplied to the corn digester was determined. Ade-
quate aeration was achieved by a single stone sparger in the 18-liter
fermentor.
More quantitative measurements of oxygen usage were made by measuring
disappearance of dissolved oxygen after interrupting aeration of a sta-
bilized fermentation. The assumption required in this method was that
the COD reduction during the period of measurement was the same as the
average COD reduction per unit of time before the aeration was inter-
rupted. A curve showing consumption of dissolved oxygen for the corn
culture by this method is represented in Figure 10. A mechanical stir-
rer was used to provide gentle agitation and homogenization of the di-
gestor components during interruption of aeration. The feed rate was
20 ml/min. of material containing 3650 mg/1 COD. The effluent assayed
204 mg/1 COD. Oxygen usage in an 18-liter fermentation was seen to be
10.2 mg/min. (0.57 mg/min./liter x 18 liters). COD reduction was 70 mg/
min. This meant that one pound of oxygen was used for every 7 pounds of
COD removed.
-34-
-------
Fig. 9a. T. viride colony
Fig. 9b. Penicillium colonies
red pigment
red pigment
Fig. 9.c. T. viride and Penicillium
Note red pigment production in area
where two generic colonies are in
close proximity.
-35-
-------
3.0
0.57 mg 02/min/l
3
MINUTES
Fig. 10. Disappearance of dissolved oxygen by T. viride growing
on corn waste in a continuous culture. The rate of oxygen disappear-
ance is shown by the slope of the line and equals 0.57 mg Oa/min/1.
-36-
-------
Amino Acid Analysis of T. viride Protein
Before undertaking feeding trials using T. viridja mycelium as the protein
source for weanling rats, it was necessary to determine the amino acid
composition of the fungal protein. Since a protein is only as good (for
feeding) as its amino acid composition and balance, any amino acid ad-
justments required to equate the protein to casein (standard diet protein)
would necessarily be determined from the amino acid study. The amino
acid composition of T_. viride protein is shown in Table 8, together with
the amino acid composition of several other good feed proteins.
Table 8
Amino Acid Composition of Several Proteins
grams/100 grams protein
Lysine
Histidine
Arginine
Cystine
Methionine
Threonine
Valine
cp-Alanine
Leu cine
Isoleucine
Tyros ine
Glycine
A Ian ine
Serine
Aspartic
Glutamic
Proline
Tryptophan
T. viride
Fungi
3.9
1.7
3.0
1.4
1.2
4.0
4.5
2.8
5.4
3.5
2.4
3.9
4.8
3.5
6.5
9.0
4.3
1.8
Corn
Normal
2.6
3.0
5.1
1.5
1.6
3.5
4.6
4.9
12.1
3.4
4.3
4.0
7.9
3.5
6.7
20.8
9.7
1.0
Opaque-2
4.2
3.5
6.8
1.7
1.4
3.3
4.9
4.4
8.4
3.2
3.9
4.8
6.5
4.3
10.0
18.7
8.6
1.3
Casein
8.0
3.0
4.0
0.2
3.4
4.8
7.7
5.9
9.8
6.5
6.5
2.5
3.0
6.5
7.0
25.0
11.0
—
Soy
Bean
6.6
2.5
7.0
1.2
1.1
3.9
5.2
4.8
7.6
5.8
3.2
3.8
4.5
5.6
8.3
18.5
5.0
1.2
Skim
Milk
8.4
2.5
3.2
0.4
2.0
4.6
6.1
4.5
9.9
10.7
6.5
2.3
—
4.3
—
22.0
—
1.2
Black and Boiling, Amino Acid Handbook, 1960 was used for the amino acid
re fe ren ce value s.
The fungal protein content = 45 percent on the basis of the amino acid
analysis.
-37-
-------
Vitamin determinations were not carried out in these studies, except for
niacin. The niacin content is 34,000 u.g/100 g of dried fungal mycelium.
This level of niacin is approximately twice that found in beef liver.
The niacin determination was performed in Dr. William Gray's laboratory
at Southern Illinois University.
Table 8 shows that the lysine content was higher than normal corn pro-
tein and equal to the high lysine corn mutant, opaque-2. It was, however,
lower than the other proteins shown in Table 8. The leucine-isoleucine
ratio was very good and equal to the casein and soy bean proteins. The
low proline value was good, since higher values are frequently associated
with poor protein quality. Threonine was higher than the corn proteins,
equal to soy protein, and slightly lower than casein. Especially notable
was the high tryptophan level. This was higher than in all other proteins
and is essential to good growth. The sulfur amino acid level was equal
to soy and skim milk. Overall, the amino acid balance was excellent.
Rat Feeding
Two diets were prepared for feeding weanling rats. Both diets contained
fat, starch, vitamins, and mineral salts optimal for weanling rat growth
as described in the section on Methods, page 14. The protein of the
standard rat diet was casein supplemented with methionine. In the test
diet, the casein was replaced with T. viride fungal mycelium collected
from the 140-day continuous corn digestion. The total protein of each
diet (casein and fungal) was 23 percent. In several instances where
there were discrepancies between the fungal protein and casein amino
acid levels, the protein (fungal and casein) were balanced with small
additions of specific L-amino acids. Both protein sources were supple-
mented with methionine to increase the S-amino acid level to that re-
quired for weanling rats.
In Figure 11 the cumulative percentage weight gains demonstrated by the
standard (casein) and test (fungal) diet-fed rats are shown during the
21-day feeding experiment. This figure shows that, after a slight ini-
tial lag, the fungus-fed rats grew at the same rate as the rats fed the
standard casein diet. The slopes of the weight gains are calculated for
each rat, and the averages show no significant differences. These are
shown in the Table insert with Figure 11. This 21-day study was a pre-
liminary examination of fungal protein characteristics when fed to ani-
mals. It would be desirable to repeat the feeding study with more rats
for a longer time period, to feed fungal protein at a low level (10 per-
cent protein), and perhaps to use other animals such as chicks and rum-
inants.
The chief purposes of this study were accomplished in that the fungal
test diet was proved palatable and digestible, and no toxic symptoms or
gross organ changes occurred. Livers, hearts, and lungs of all the rats
were examined and weighed at the conclusion of the feeding trial. No
noteworthy gross changes were observed in weight or appearance in any of
the organs.
-38-
-------
150
o
•o
E
3 |25
z
o
i IOO
ED
fi 75
o
a:
UJ
a.
ui
r 50
" 25
-25
I I I
«*^»
SLOPE OF WEIGHT GAIN
STANDARD
DIET
X X
FUNGAL
DIET
• •
/RAT
6.7
6.2
7.4
6.9
6.7
6.6
AVG.
6.76
6.73
I I 1
8
DAYS
10
12
14
16
18
20
Fig. 11. Rat growth curves.
-------
Digestibility and net protein utilization (NPU) were based on nitrogen
analyses of the animal feeds, urine, and fecal samples collected sepa-
rately each day during the rat feeding experiment. These are apparent
digestibility and NPU values, since no endogenous nitrogen was consid-
ered. Thus, on the basis of diets containing 23 percent protein, the
apparent digestibility for the standard casein diet was 97 percent and
for the fungal diet, 90 percent. These values were determined from the
standard nutritional formula of
IN - FN
-5r-
where IN is the intake nitrogen and FN the fecal nitrogen. The apparent
net protein utilization (NPU) was also determined from the equation:
IN
where IN and FN are as described above, and UN is the urinary nitrogen.
The results indicated an NPU for the standard casein diet of 75 percent
and 50 percent for the fungal (test) diet. The 50 percent net utiliza-
tion of the fungal protein is undoubtedly a low estimate, since no cor-
rection has been made for the fact that about 20 percent of the nitrogen
of the mycelium is non-protein-nitrogen.
Soy Whey; HC1 Soy Whey
Fungal Strain Selection
Initial selection of fungal strains for application to soy wastes was
made with attention to rate and final level of COD reduction and in-
crease in mycelial mass (fungal growth). Adaptation of the organism to
the substrate was done before making judgments on the potentialities of
the strain. This was done by making initial transfers through sterilized
soy wastes at 24-hour intervals. In these initial strain selection exper-
iments, no adjustments of pH or additions of nutrients were attempted.
The pH of the soy whey, as received, was 4.5. Soy whey used in these
studies was obtained from a process in which hydrochloric acid had been
used for protein precipitation. Results are shown in Table 9.
The supernatants from these fermentations were transparent but yellow-
brown in color except in the cases of P_. elegans 1-134 and T. viride
1-185. In these two instances, the supernatant was clear and colorless.
Most of the fungi produced pellet type growth, but the highly efficient
!• viride 1-185 strain (76 percent COD reduction in 24 hours) produced
a heavy filamentous matted mycelium.
The rate of COD reduction was examined in greater detail using the three
cultures that appeared most promising in these screening studies. These
were T. viride 1-185, T. viride 1-187, and P. elegans 1-134. Both 1-187
and 1-134 grew more slowly than 1-185 and became heavily contaminated
with organisms of the natural biota before maximum COD reduction was at-
tained. Thus, it would appear from this survey that T. viride 1-185 was
the strain of choice for additional work on the HC1 soy wheys.
-40-
-------
Table 9
Digestion of Soy Waste by Various Species
and Strains of Fungi Imperfect!
Fungi
Natural biota
Trichoderma viride 1-23
Trichodertna viride 1-23
Trichoderma viride 1-184
Trichoderma viride 1-186
Trichoderma viride 1-188
Trichoderma viride 1-192
Trichoderma viricte 1-193
Trichoderma viride 1-191
Trichoderma viride 1-190
Trichoderma virj.de M-114
Trichoderma viride 1-187
Trichoderma viride 1-185
*Tricho derma viride 1-185
Gliocladium deliquescens 1-31
Gliocladium deliquescens 1-31
Paecilomyces ej.egans 1-134
Paecilomyces elegans 1-134
COD mg/1
0 hr
7429
7429
7600
7600
7600
7600
7600
7600
7600
7600
7600
7600
7600
7600
7429
7600'
7429
7600
24 hr
6315
3450
4300
4300
4300
4300
4150
3925
3900
3850
2800
2475
1825
1125
2900
2825
3750
1800
COD
Percentage
Reduction
15
54
43
43
43
43
46
48
49
50
64
68
76
86
61
63
50
77
Carbohydrate mg/1
0 hr
3988
3988
3988
3988
24 hr
2970-
2410
1880
1875
72 hr
1940
1095
1000
531
Sterilized soy waste used in this instance.
pH Effects
It is known that many fungi require acid environments for best performance,
Cochrane, (23). Further, one would expect many of the competing
organisms to be inhibited at low pH values. Studies of pH effects were
conducted with T_. viride 1-185 using 30 ml samples of soy whey in shake
flasks. pH adjustments were made with 1 N HC1 and 1 N NaOH. Incubation
was at 27°C for 24 hours. The optimal pH appeared to lie between 3 and 4
as shown in Figure 12. During the fermentation of soy whey, the pH ini-
tially dropped from 4.5 to 3.2-3.5. After 24 hours, unless acid additions
were made to these flasks, the pH rose to 7.0.
-41-
-------
g
100
90
80
70
60
§ 5°
«P 40
o
« 30
P-4
20
10
0
PH
Fig. 12. Effect of pH on COD reduction by
T. viride 185 in soy whey after 24 hours.
-42-
-------
Temperature Effects
Temperature effects were also studied with T. viride 1-185 in 30 ml
shake flask cultures. The pH of the soy waste was maintained at 3.5
in these studies. Results are shown in Figure 13. The optimal tem-
perature for rapid COD reduction appeared to lie between 27°C and
35°C.
Effects of Inoculum Size
Because of the high levels of carbohydrate and protein contained in soy
whey it was difficult to establish T_. viride as the predominating orga-
nism. It was reasoned that a large inoculum should increase the effici-
ency of conversion of COD to mycelial mass by overwhelming "contaminating"
organisms, s'hould increase the rate of COD reduction and should aid the
fungus to establish itself in the nonsterile soy whey. These effects
were examined in experiments in which various size inocula of T_. viride
1-185 were added to 30 ml shake flask cultures of raw soy whey. The soy
whey contained 10,920 mg COD/1 and was diluted by the inoculum to about
9800 COD mg/1. Results are shown in Table 10. The increased fungal
mass was seen to be relatively independent of inoculum size over a nine-
fold range of inocula. Mycelial mass attained a constant level in every
case after 24 hours. Thus, it would appear that the fungus can compete
with the "contaminating" organisms in the soy whey at even the lowest
inoculum level during the first 24 hours.
Table 10
Increase in Mass of T., viride 1-185 Mycelium
Produced as a Function of Inoculum Size
T » Fungal mass
Inoculum . . „, ,
, - increase in 24 hr
mg/ml mg/ml
0.5 2.3
1.3 2.4
2.3 2.4
4.5 2.0
The details of the soy digestion were examined in greater depth. In
Figure 14 it is seen that the rate of COD reduction was initially greater
when larger inocula were used. All fermentations, however, reached about
the same COD level after 24 hours. The progression of initial COD diges-
tion rates with inoculum size is plotted in Figure 15. The efficiency of
conversion of COD to mycelial mass in these particular fermentations was
about 40 percent on a weight basis; that is approximately 40 mg of my-
celium was produced for each 100 mg of COD utilized.
-43-
-------
100
90
80
70
§ 60
£
I
e 50
O)
60
2 40
§
S-l
S 30
20
10
I I I I
10 15 20 25 30 35
Temperature (°C)
40 45 50
Fig. 13. Effect of temperature on COD reduction by
31s. viride 185 in soy whey after 24 hours.
-44-
-------
10
8
tf
4.5
8 12 16
Ti«e (Hours)
20
24
Fig. 14. COD reduction as a function of
inoculum size.
234
laoculu* Sise
Fig. 15. Kate of COD reduction as a
function of inoculua size. The early linear
rates (8 hr«) in Fig- 14 above were u«ed for
the curve in Fig.. 15v
-45-
-------
Nutrient Addition
The failure of these fungal strains to bring COD levels to lower values
in reasonable fermentation times prompted exploration of nutritional fac-
tors which might have been limiting fungal metabolism. As a first step,
the chemical composition of the original soy whey and the filtrate after
growth of T_. viride 1-185 on the soy waste for 48 hours were examined.
Results are tabulated in Table 11.
Table 11
Chemical Analyses of Soy Waste Before and
After 48 Hours' Digestion by T. yiride 1-185
Test
COD
BOD
Nitrogen (Kjeldahl)
NPN (nonprotein nitrogen)
Protein
Carbohydrate
Chlorides
Nitrates (as N)
Nitrites (as N)
Total Phosphate
Total Soluble P04
Ortho Phosphate
Total Solids'
Ash (residual solids)
pH (units)
Before Fungal
Digestion
mg/1
7800
5420
600
102
3013
3980
653
7
0.01
87
82
78
8300
1590
4.6
After Fungal
Digestion
mg/1
1800
860
96.7
71.5
156
800
557
5
0.13
45.6
32.5
29.0
4170
1130
7.4
Percentage
Reduction
77
84
84
30
95
80
15
28
—
48
60
63
50
29
Analyses performed 0n samples of the effluent were made after filtering
through a single layer of Whatman No. 4 filter paper.
The results indicated that the protein nitrogen had been disproportionately
depleted, as compared to carbohydrates. The nonprotein nitrogen was used
to a much lower degree. These findings perhaps indicated that the" reduction
of BOD may have been limited by the available nitrogen supply. Trials of
the effect of nitrogen additions were therefore conducted in shake flask
cultures of T?. viride on raw soy whey. Considerably greater, but still in-
complete, removal of BOD was achieved when nitrogen was added as ammonium
sulfate. These results are shown in Table 12. No further reduction of COD
or BOD was achieved with higher levels of nitrogen.
-46-
-------
Table 12
Effect of Nitrogen Supplementation on Fungal Digestion of Soy Whey
Fungus
None
T. viride
G. deliquescens
pH
3.8
3.2
3.5
COD mg/1
None
0 hr
9,870
10,100
10,020
48 hr
8,870
2,530
2,310
Nitrogen
0 hr
9,900
9,800
9,900
48 hr
8,100
1 ,660
1,000
BOD mg/1
None
0 hr
6,200
5,850
5,420
48 hr
6,180
940
869
Nitrogen
0 hr
6,100
5,620
5,860
48 hr
5,940
560
340
Nitrogen added as (NH4)2S04 at a final concentration of 0.01 molar.
Residual COD
Besides nutrient depletion, other hypotheses accounting for incomplete re-
moval of COD and BOD were formulated and tested. One hypothesis was that an
inhibitor of further metabolism accumulated during soy digestion with these
fungi. One test of this hypothesis was based on the assumption that a dia-
lyzable inhibitor was formed. Spent soy whey was dialyzed for 16 hours, re-
constituted by addition of ammonium sulfate and phosphate ions and reinocu-
lated with various fungal strains. No further reductions in COD levels were
achieved. The dialysis had been sufficiently extensive to remove half the
residual COD. Thus, the inhibitor, if such existed, was not a small molecule,
Another test of the possible accumulation of an inhibitor substance was con-
ducted by examining the amount of growth and COD reduction at a series of
soy whey dilutions. The rationale was that the dilution of the soy whey
would allow consumption of a greater proportion of the COD before an inhibi-
tor attained critical concentrations. This would be in contrast with the
findings to be expected if metabolism was limited by exhaustion of an essen-
tial nutrient. In this latter case, one would expect the same percentage
removal of COD before slowing of the COD removal, regardless of the original
COD concentration.
Such a study was conducted using dilutions of soy whey ranging from 10,000
to 2500 mg COD/1. Shake flasks (125 ml) were inoculated with 2 mg/ml of
T_. viride 1-185 and sampled at 0, 8, 16, and 24 hours. At 16 hours the
COD removal had reached maximum with the residual values as shown in Table
13.
The fact that a nearly constant proportion of the COD was used before the
reduction of COD halted may be taken as evidence of nutrient depletion and
as evidence against the accumulation of an inhibitor to critical growth-
limiting levels. This line of reasoning presupposes that the amount of in-
hibitor produced would be a function of the amount of COD utilized and that
its effectiveness would be a function of its concentration per unit volume.
-47-
-------
Table 13
Residual COD as a Function of Dilution
of Soy Whey before Inoculation
Original
COD mg/1
9850
8350
5800
4400
2500
After 16 Hours Incubation
COD mg/1
2900
2410
1700
1000
850
Percent of Original COD
30
29
29
23
34
Still another approach to the removal of residual COD was to inoculate
a spent liquor from one fermentation with other fungal strains. A liquor
from a 16-hour fermentation with T_. yiride 1-185 was filtered, adjusted
to pH 4.2, and reinoculated with a variety of fungi. Of eighteen strains
tested, only seven were effective in further reducing the COD (Figure 16).
Four strains gave essentially identical results and are plotted on a
single curve. No significant reduction of the residual COD level was
observed.
A related approach was to inoculate simultaneously with two fungal strains.
This was done using both the original undigested soy whey and the residual
whey after an initial 24-hour digestion by T. yiride. It was hoped that
the metabolic capabilities of two strains might prove complementary and
so permit more nearly complete digestion that could be attained by any one
strain alone. Combinations used included T. viride 1-185 with Gliocladium
deliquescens, T_. viride 1-185 with Aspergillus oryzae, G. deliquescens and
A. oryzae, T_. viride 1-192 with £. deliquescens, and T_. viride 1-192 with
A. Qjryzae. None of these fungal combinations reduced the COD below
approximately 2000 mg/1.
The most direct experimental evidence that residual COD was not inhibitory
to fungal metabolism was shown in an experiment where cellulose was added
to the soy whey supernatant. The supernatant was the filtrate from a 48
hour fungal digestion of raw soy whey. It was reasoned that any metabolic
inhibition contained in the digested soy whey would block the utilization
of a prime energy source (cellulose) for this fungus. The data in Table
14 show that no inhibition of cellulose was present. All added cellulose
was digested and apparently stimulated further reduction of the residual
soy whey. This is shown in Table 14 where the COD of 1405 (without cellu-
lose)was reduced to 1325.
-48-
-------
3000
(30
s
§
o
2500
2000
1500
1000
I
T
191
186
188
193
T. viride-192
T. viride— 185
deliquescens<
31
1. T. viride1
2.
3.
4. G
-O 1.
16
24
Hours
32
48
Fig. 16. Fungal growth in soy whey predigested for 16 hours with T. viride 185.
-49-
-------
Table 14
Digestion of Spent Soy Supernatant by G. deliquescens
No.
1
2
3
4
5
Sample
Soy whey
Soy whey
Soy whey
Supernatant
Soy whey
Supernatant
Soy whey
Supernatant
Fungus
mg
0
60
60
0
60
Cellulose
mg
0
0
0
30
30
Hours
0
48
48
0
48
COD
mg/1
10,800
2,663
1,405
3,683
1,325
Experimental Conditions:
Soy Whey 30 ml cultures in 125 ml Erlenmeyer flasks incubated for
48 hours at 26°C in a rotary shaker at 140 ro tat ions/tnin.
Soy Whey Supernatant 30 ml filtrate after 48 hr soy whey digestion
by G. deliquescens. Reinoculated with fungus and incubated an
additional 48 hours, as above, with and without cellulose.
It appeared from these experiments that the accumulation of inhibitors
did not account for the incomplete digestion observed. Complete diges-
tion of this remaining substrate must await additional studies designed
to reveal whether or not nutritional imbalances exist, and the chemical
character of the residual materials. It is entirely possible that cer-
tain carbohydrates and proteins in soy whey are refractory to further
fungal metabolism. That nutritional imbalances do develop was learned
earlier where at least one, the available utilizable nitrogen, was re-
quired for further fungal digestion (Table 12). There was no stimula-
tion by additional phosphate as was observed in the corn waste studies.
This was not surprising, since 45 mg phosphate/1 was found in the resid-
ual soy whey filtrates.
Continuous Fermentation
Continuous fermentation of HC1 precipitated soy whey was tried in a
second fermentor constructed similarly to the one used for corn waste
digestion. A diagram of the apparatus was shown and described in
Figure 1, page 12.
Initially, the continuous soy fermentation systems were started as "batch"
type in order to establish a heavy culture of fungal mycelium before a
continuous flow of soy whey was introduced. Thus, a typical experiment
selected from these early attempts to establish a continuous digestion
of soy whey was run as follows:
-50-
-------
A 24-hour culture of Gliocladium deliquescens grown in sterile HCL soy
whey was used for the inoculum. The raw soy whey contained 11,820 mg
COD/1. 4.7 liters of this fungal culture containing 21 gm of fungus
(dry weight) were added to 11.3 liters of freshly thawed soy whey.
Thus, an initial digestor volume of 16 liters contained 1.3 g fungus and
8400 mg COD/liter. At zero time, the pH was 4.0 and the total suspended
solids (dry weight) were 13,500 mg/1, of which 1300 mg were fungus and
12,200 mg were say particulates. Aeration and mixing were accomplished
with 3 stone spargers which supplied a dissolved oxygen concentration
of never less than 0.5 mg Og/l.
The operation of this digestion system began as a batch process as shown
in Figure 17 where COD reduction is plotted vs. time in days. After 65
hours, the batch digestion was switched to continuous, and the flow rate
of fresh raw soy medium was started at 2.5 ml/min. This feed rate cor-
responded to a soy influx of 0.15 1/hr and supplied 2150 mg COD/hr. At
this feed rate there would be a complete digestor turnover every 100
hours. At zero time of continuous digestion (zero + 65 hr), the fungus
appeared mature with some budding and a few lysed mycelial fragments.
Large swollen budding mycelium, indicative of young, actively metaboliz-
ing organisms, was absent. Bacteria were present in very high numbers.
We concluded that the culture was too aged for an efficient continuous
soy digestion. However, when the feed rate was increased to 4 ml/min.
(60 hr digestor turnover time), the fungus developed new growing tips
and maintained a stable microscopic morphological appearance. The
fungal stability was also demonstrated by the continuous decrease in
the COD which leveled off at approximately 1600 tng/liter after the
eighth day and continued at this level through the twelfth day.
When the COD was not reduced below 1500 mg/1 after thirteen days, the
flow of soy whey was shut off and the system reverted to a "batch" fer-
mentation for twenty hours. It was thought that by reverting to batch
operation, the residual COD would be further reduced. However, the COD
increased to 2500 mg/1 because of fungal starvation and lysis. Yeast
and bacterial contamination increased to a high level (approximately 106
organisms/ml). Therefore, on the fifteenth day, the system was switched
from batch back to continuous, and the feeding was set at 5 ml/min. (not
shown in Figure 17). The raw feed was changed from HC1 soy whey to SOg
soy whey (the S03 was adjusted to 200 mg/1). This change to S02 soy
whey was made with the hope that the SOs would reduce the yeast and bac-
terial contamination. Neither of these changes (batch of S02) resulted
in good COD reduction. Although the mycelial dry weight increased from
2.5 g/1 at the twelfth day to 3.5 g/1 on the fifteenth day, and remained
at the 3.5 g/1 level during the SOs soy whey feeding, the COD remained
approximately 3000 mg/1. Although the COD of the incoming SOs soy whey
was reduced during this period of SOg feeding from 13,000 mg/1 to 3000
mg/1, no further reduction in COD resulted through the 23rd day. The
fungal appearance was that of an old, stationary phase culture, and
sporulation and lysis were beginning. Since this was a sign of starva-
tion, the feed rate was increased to 10 ml/min. The increased feed rate
did not stimulate new fungal growth but increased the bacterial and yeast
contamination. Thus, it was believed the "aged" fungal culture was los-
ing out to the contaminants. The experiment was therefore discontinued.
-51-
-------
8000
7000 ~
6000 -
5000 -
8 4000
3000 -
2000 -
1000 -
1 1 1 1
Continuous 2.5 ml/min
11 12
Fig. 17- Continuous COD reduction of HCl soy whey by G^ deliquescens.
-52-
-------
Inoculation-Dilution. When the previous problems with the continuous
soy digestion system were described to Dr. H. Orin Halvorson, University
of Minnesota, he made several suggestions. One suggestion was with re-
gard to initiating the continuous culture system and avoiding an early
build-up of other microorganisms indigenous to the soy whey (yeasts and
bacteria). This technique was the inoculation-dilution procedure.
This technique consisted of introducing a small amount of soy whey into
a fermentor nearly filled with water. A fungal inoculum appropriate to
the amount of soy whey was introduced, and continuous feeding of the fer-
mentation with fresh soy whey began at zero time. This technique precludes
the presence of much larger amounts of nutrient than can be quickly uti-
lized by the mycelial mass present and which might otherwise support the
growth of contaminants. The mycelial mass grows as incoming soy whey
gradually replaces the water initially present. The advantages of this
procedure for large-scale start-up operations are obvious.
One such experiment is illustrated in Figure 18. In this experiment
(Figure 18), 750 ml of HC1 soy whey nutrient + 1.2 g of wet Trichoderma
viride 1-185 mycelium were diluted in the digester jug with 14,250 ml
of tap water. The raw soy feed (full strength) was turned on at zero
time, at a feed rate of 5 ml/min. Figure 18 shows the theoretical time
required for the soy concentration to reach 100 percent. The "actual"
curve (COD assay) is compared to the "theoretical" COD curve. The
"theoretical" curve represents the increasing concentration of raw soy
COD, and the "actual" represents the impact of the fungal growth and
COD reduction on the "theoretical".
Although the technique of inoculation-dilution was successful and the T.
viride culture reduced COD for a few days, it soon showed microscopic
evidence of deterioration and gave poor COD removal. This pattern was
repeated on several trials. One difficulty appeared to be "bulking".
The T. viride mycelium gathered into floating masses rather than being
dispersed through the medium. Stirring was only partially successful in
overcoming this difficulty. Attention was therefore turned to another
organism which had given relatively good behavior in earlier trials and
did not show the clumping tendency. This was Gliocladium deliquescens.
Although this organism did not give difficulties with bulking, difficul-
ties were still encountered in maintaining a stable fermentation. Fer-
mentations began well enough but soon deteriorated, and competing orga-
nisms began to appear. It was noted from microscopic observations that
the organisms showed vacuolation and sporulation before competing orga-
nisms began to predominate. This suggested that the fungus had begun to
starve and go into a stationary phase with some lysis. When this occurred,
competing organisms obtained a foothold. Once in a stationary phase, the
fungus responded only slowly to adequate nutrient concentrations.
Two expedients were evident as ways to prevent starvation. One was simply ,
to regulate the feed rate by COD measurements in the fermentation so that
there would always be adequate nutrient present. Of course, too great an
excess of nutrient (high feed rate) would mean an outflow stream of high
-53-
-------
o>
E
o
"5
a>
o
o
o
o
II
>.
o
CO
10,320
10,000
9,000
8,000
7,000
epoo
5,000
4,000
3,000
2,000
1,000
0
I 1 1 1 1
,^^~~~"^~~-
l/'
<^— Theoretical COD
/
/
/
/
/
/
/
lc
!£ at t=0, COD = 5l6mg/l =C0
lM2hr
' /
- /
/26%
I Px.,,67% /Actual COD
/ ^v?T/o
• i i i i
0 40 80 120 160 200 240
Hours
Fig. 18. Inoculation-dilution technique for starting a
continuous digestion of soy whey by T. viride. Determination
of the "theoretical" COD was according to the integrated
formula c_Coo _ £
c5=c3b= e v
-54-
-------
COD and a washout of fungal mycelium. The other expedient (suggested by
Dr. H. Orin Halvorson) was frequent removal of fungal mass while keeping
the feed rate constant. The chief criterion in this case is the weight
of fungal mass allowed to remain in the fermentation per unit volume.
This is simply another way of assuring that there is not an excess of
mycelium for the incoming nutrient.
Fungal Mass Control. A study of continuous soy whey digestion by em-
ploying fungal mass control is shown in Figure 19. This experiment was
done using G. deliquescens. Inoculation was at the level of 3.2 g of
young (24 hour) mycelium to 16 1 of tap water. Feed with soy whey was
at the rate of 6 ml/min. into an 18-liter fermentor.
The data shown in Figure 19 are divided for purposes of explanation into
three areas (A,B,C). In Area A the fungal mass was increasing as the COD
feed concentration was increasing (refer to theoretical plot). As the
fungal mass increased, the actual COD concentration was maintained at a
near steady state of approximately 1500 mg/1. At the five-day point,
when the fungal mass was 2.8 gm/1 and the COD had reached 1250 mg/1 the
decision was made, on the basis of microscopic examination of the fungus,
to remove an aliquot of the digestion mixture, separate the fungus,.and
return the supernatant liquor to the fermentation jug. This removal of
part of the fungal mass accounted for the decrement in mass seen in the
latter part of Area A of Figure 19. Before the effectiveness of the
fungal mass removal could be assessed, the feed supply system clogged.
Because of feed (COD) deficiency, fungal starvation occurred over the
next 24 hours (sixth to seventh day in Figure 19) and fungal cytoplasmic
material was spilled into the fermentor, causing a rapid COD increase
(Area B) and a heavy growth of yeast and bacteria. Between days 7 and 8
the entire fungal mass was removed from the fermentor, washed (thus re-
moving yeast and bacteria), and the fungal mass returned to the fermentor
and a fresh soy whey, adjusted to a COD of 4500 mg/1, was added. The
feed flow was started again at a rate of 5 ml/min. It can be observed
(Figure 19, Area B) that, as the fungal mass increased, the COD decreased
until the sixteenth day, where a fungal mass of 2 g/1 reduced the COD to
2000 mg/1 at a flow rate of 5 ml/min. In Area C, physical removal of
fungi to maintain a level of 3.3 g/1 was done about every third day, and
a consistent COD level was maintained between 1300 and 1500 mg/1 for
approximately twelve days. We believed this state could have been main-
tained indefinitely. Had there not been a feed block on the sixth and
seventh days, the system might well have maintained the state observed
over the last twelve days throughout the thirty-day period. The arrows
in Area C, Figure 19, indicate the points at which the fungal removal
technique was used.
The residual COD level of between 1100 and 1600 mg/1 appeared to be
material not preferentially metabolized in this system. In Area C we
were not successful in getting the COD substantially below 1100 mg/1
in this continuous culture. Although a residual COD of 1500 to 1800
mg/1 was maintained at a flow rate of 7.5 ml/min. (not shown in Figure
19), feeding at 10 ml/min. resulted in a COD of 2500 mg/1 and higher.
-55-
-------
i 1,000
7,000
6,000
5,000
o
o
o
4,000
3,000
2,000
1,000
THEORETICAL
COD
Removed fungal mass
I
Removed
fungal
mass
CO
- 2 u-
30
Fig, 19 . Continuous digestion of soy whey by C. deliquescent. Effect
of controlling fungal mass, on stability of COD digestion. The theoretical
COD is shown by the bent arrow, the fungal dry weight as 0 0 and the
actual COD as • •.
-56-
-------
Because further examination of these parameters could be studied with
SQs soy whey as well as with the HC1 soy whey and because the major
amount of industrial soy processing is done with SOg , it was concluded
that further continuous culture studies should be done on SOg soy wheys.
Dissolved Oxygen Utilization
Oxygen disappearance during continuous digestion of HC1 soy whey by G_.
deliquescens was measured immediately after interruption of aeration.
The technique and assumptions for taking these data were the same as
those used earlier for oxygen utilization in the corn waste digestion
system. The data are plotted in Figure 20.
Making the same calculations for the soy fermentation as was done for
corn in a 15-liter fermentation, we arrived at an estimate of 5.5 pounds
of COD removed per pound of oxygen used. (The incoming feed contained
10,320 mg/1 COD and the effluent contained 2600 mg/1. The feed rate was
4 ml/min. and the fermentor contained 15 liters total volume.)
Atnino Acid Analysis
The amino acid analysis of the fungal strain used for the HC1 soy whey
digestion study is shown in Table 15. The analysis for G. deliquescens
is compared to earlier analyses done on T_. viride and to several other
proteins. Especially significant are the values for lysine, threonine
and tryptophan, which are 6.15, 4.86, and 2.31 g/100 g protein, respec-
tively. This G. deliquescens amino acid analysis was considered valu-
able for formulation of diets for animal feeding trials.
-57-
-------
3.0
2.5
o>
£
2.0
UJ
o
g
Q
liJ
1.5
o
CO
«2 1.0
0!
0.33 mg 02/min/l
3
MINUTES
Fig. 20. Disappearance of dissolved oxygen by G. deliquescens
growing on soy whey in continuous culture. The rate of oxygen dis-
appearance is shown by the slope of the line and equals 0.33 mg Oa/
rain/1.
-58-
-------
Table 15
Amino Acid Analysis of Two Fungal Strains Compared to
Several Standard Proteins (grams/100 grams protein)
Amino Acid
Lysine
Histidine
Arginine
Aspartic Acid
Threonine
Serine
Glutamic Acid
Pro line
Glycine
Alanine
Cystine
Methionine
Valine
Isoleucine
Leucine
Tyros ine
Phenylalanine
Tryptophan
Trichoderma
viride
3.94
1.67
2.98
6.49
3.94
3.51
8.98
4.34
3.88
4.76
1.38
1.20
4.48
3.52
5.35
2.44
2.76
1.80
Gliocladium
deliquescens
6.15
2.33
5.17
8.41
4.86
4.71
9.47
4.25
4.39
5.99
1.42
1.19
4.85
4.06
6.18
3.29
3.96
2.31
Casein
8.0
3.0
4.0
7.0
4.7
6.7
25.0
11.0
2.5
3.0
1.0
3.5
7.7
6.5
9.7
6.5
5.9
1.2
Soy Bean
Meal
6.6
2.5
7.0
8.3
3.9
5.6
18.5
5.0
3.8
4.5
1.2
1.1
5.2
5.8
7.6
3.2
4.8
1.2
Opaque-2
Corn
4.2
3.5
6.8
10.0
3.3
4.3
18.7
8.6
4.8
6.5
1.7
1.4
4.9
3.2
8.4
3.9
4.4
1.3
The protein content of these fungal mycelia is between 42 and 45 percent,
based on the amino acid analyses.
Soy Whey; S02 Soy Whey
Soy wheys from commercial processes in which the soy protein was precipi-
tated by sulfur dioxide were found to present separate problems in that
the sulfur dioxide markedly inhibited fungal growth.
Fungal Strain Selection
Since high concentrations of sulfur dioxide were markedly inhibitory,
initial studies were conducted with a soy whey which contained 147 tng/1
of sulfur dioxide. Growth of several fungus strains on this S02 soy
whey medium are shown in Table 16.
-59-
-------
Table 16
24-Hour Reduction of COD and S02 in a
Soy Whey Containing 147 mg SOy/1
Fungi
None
Natural biota
T. viride strain 1-185
T. viride strain 1-190
T. viride strain 1-192
T. viride strain 1-23
G. deliquescens strain 1-31
A. oryzae strain 1-14
Final Values
COD
mg/1
12,800
12,200
12,330
4,357
5 , 74ff
3,170
2,110
1,750
S02
mg/1
147.0
140.0
13.0
20.0
6.4
6.4
6.4
<6.4
The more successful strains (Table 16) were seen to have reduced markedly
both the COD and sulfur dioxide levels. The most promising strains were
then tested in soy whey containing a COD of 12,030 mg/1 and 415 mg/1 of
sulfur dioxide. This was attained by mixing 1 part of a soy whey contain-
ing 1203 mg S02/l with 3 parts of a whey containing 147 mg S02/l. Results
are plotted in Figure 21. A. oryzae 1-14 was the most effective fungus of
the group to reduce COD in the presence of 415 mg S02/l of soy whey.
Studies with A. oryzae at a variety of S02 concentrations are shown in
Figure 22. There was little difference in the rates of digestion at con-
centrations of sulfur dioxide up to 513 mg/1.
Substrain Selection for Rapid S02 Removal
Greater tolerance of sulfur dioxide than 513 mg S02/l was required to
handle several of the waste streams from commercial plants. Attempts
were therefore made to select more effective fungal substrains by serial
passage of A. oryzae through a soy whey containing 710 mg S02/l. This
was a concentration which originally allowed only very slow COD utiliza-
tion (Figure 22). Results of these serial transfers are shown in Figure
23. It is seen that repeated passage yielded a much more rapid growing
culture at this sulfur dioxide concentration.
Extension of these techniques developed fungal cultures which grew at S02
concentrations up to 900 mg S02/l. Similar studies with G. deliquescens
grown in S02 also showed similar results to those described for A. oryzae.
-60-
-------
12,000
10,000
8,000
|»6,000
§
4,000 -
2,000 -
12
24
36
Boors
48
60
72
Fig. 21. Activity of fungi on GOD reduction of SOa »Oy whey
containing 415 mg SOa/1.
-61-
-------
12,000
10,000 -
i.OOO -
M 6,000
>.x
§
4,000 -
2,000 -
513 ag SOa/1
415 mg SOs/1
Fig. 22. COD reduction of SOa soy whey by A. oryzae.
-62-
-------
12,000
10,000 -
8,000 -
9 6,000 -
>«^ '
Q
3
Fig. 23. Effect of rapid passage ef A^ oryzae on COD reduction of
S0» containing soy whey at 710 mg SOa/1.
-63-
-------
The efficacy of a fungus adapted to grow at 500 mg S02/l was tested at
a series of sulfur dioxide concentrations. Results are shown in Table 17,
Table 17
Growth of an A. oryzae Adapted to Sulfur Dioxide
at Varying Sulfur Dioxide Concentrations
S02
mg/1
0
95
157
269
529
788
COD Reduction
24 hours
mg/1
3160
4690
4710
5870
4690
2900
Fungal Mass
mg/1
1780
2300
2880
3020
3340
2680
Ratio
mg Fungal Mass
mg COD used
0.56
0.49
0.52
0.52
0.73
0.91
It was observed that the rate of COD utilization and the rate of accumu-
lation of fungal mass was actually greater in the presence of S02 than
in its absence. S02 inhibition was observed again at the highest levels
of sulfur dioxide. It was also of interest, but must be considered as
preliminary evidence, that the efficiency of conversion of COD to fungal
mass was highest at the highest sulfur dioxide concentration (Table 17).
Experiments were conducted on the utilization of sulfur dioxide by rapidly
transferred (adapted and nonadapted) A. oryzae at increasing levels of sul-
fur dioxide. "Adapted" strains were passed several times through soy medium
containing sulfur dioxide. S02 measurements were made after 4 hours of
incubation (Figure 24). The "adapted" (S02 pregrown) organisms showed
nearly complete utilization of S02 at the lower levels of sulfur dioxide
in the four hours incubation. The "nonadapted" (not S02 pregrown) showed
a rate of S02 utilization equal to that observed for the "adapted" strain.
Both curves (Figure 24) appeared to be reaching a common S02 level at ap-
proximately 900 mg S02/l. Both fungal strains appeared to have undergone
S02 induction.
Two questions that arose as a result of these studies with the non-S02-
pregrown fungal strain were: (1) was the fungal mycelium undergoing en-
zyme induction at the lower S02 levels which enabled it to remove (utilize)
the S02? (2) was this the same induction process that had occurred in the
fungal mycelium during pregrowth on S02? We rejected the hypothesis that
fungal adsorption of S02 could explain the utilization (uptake) of S02.
If this were true, the S02 utilized by both the "adapted" and "nonadapted"
fungi (Figure 24) would show similar S02 uptake rates from zero S02 con-
centration.
-64-
-------
500
Theoretical line for
complete utlllcct
Bon-SOa-pregrovn
200
400 600
I»itial SO* '(
800
1000
Fig. 24. Utilization of 80s by A. ory«ae pregrovn in
the presence end absence of SOa.
-65-
-------
Continuous Fermentation
The inoculum was strain 1-14 of A. oryzae obtained from Dr. William Gray.
This continuous culture was also initiated by the inoculation-dilution
method described for the HC1 soy experiments. The A. oryzae was grown
in sterile S02 soy whey prior to inoculating the continuous culture di-
gestor. Transfers to fresh medium during the pre-continuous culture
stage were made at gradually shorter time intervals in shake culture
flasks (batch). Higher concentrations of S02 were employed until a
rapid growing and S02 utilizing variant was selected for the continu-
ous culture experiment.
Although this strain of A. oryzae was capable of rapidly removing S02
and had been selected for these S02 wheys, the COD reduction by this
fungal strain in the continuous fermentation was too slow and less com-
plete (residual COD of 3000 mg/1) than that observed in batch culture.
Therefore, we proceeded to establish another continuous fermentation
with G_. deliquescens. This organism had also shown many desirable fea-
tures in earlier batch screening investigations. In addition, if this
fungal strain was satisfactory in continuous digestion, animal feeding
experiments could be initiated with a fungal mycelium which had an ex-
cellent amino acid composition (Table 15) and of which we had collected
a substantial amount of dried mycelium.
The G_. deliquescens was removed from stock neopeptone —dextrose agar
slants and used to inoculate sterile S02 soy whey at a level of 200 mg
S02/l and 10,500 mg COD/1. The mycelium was transferred rapidly several
times through this medium, and a 50-percent inoculum was prepared by add-
ing the fungus from 500 ml of this medium to 1000 ml of fresh S02 soy
whey. The one liter of inoculated soy whey was diluted by 15 liters of
tap water in the continuous fermentor as previously described for our
now standard inoculation-dilution procedure. The feed rate was set to
deliver raw S02 soy whey at a rate of 5 ml/min. The results of this
experiment are shown in Figure 25.
The theoretical COD was plotted to indicate the rate of COD build-up in
the absence of the fungus. The data show that the critical level of
actively metabolizing mycelium at a flow rate of 5 ml/min. was approx-
imately 3.2 to 3.5 g/1. The microscopic data also confirmed the data
shown here. Microscopically, at any fungal mass level above 3.5 g/1,
the mycelium developed long, thin, granular strands and lysed. At the
point (arrow) described as "equilibrium", optimal metabolism of the soy
waste was achieved (Figure 25). Control of the fungal mass by physic-
ally removing mycelium at the 3.5 g/1 level possibly would have main-
tained a constant COD residual level. Since this was not done, the
fungus sporulated, lysed (signs of starvation), and yeasts and bacteria
took over.
Fungal Mass Control. The entire procedure for preparing an inoculum,
transferring rapidly through increasing S02 concentrations in soy whey
sterile medium, and finally, inoculating the fermentor with the prepared
G. deliquescens 1-31 strain via the inoculation-dilution route was re-
peated as in previous experiments.
-66-
-------
11,000
^
8,000
^6,000
O»
E
*^— ^*
p
8 4,000
2,000
5
0
/ ^
^ /^
/THEORETICAL
y COD
X
/.
/
/
/ /EQUILIBRIUM o
~\ 1 i
\ * /
' / /
/ / /
/ / /ACTUAL
/of y° COD
jO0 ^^.^-O^
/O °
i i i i
4
rH
3 "J
CO
C
0)
o
CO
o
3
2 5
0)
•a
cSl
1
0
02468
DAYS
Fig. 25. Continuous digestion of SOa soy whey
by G. deliquescens. The theoretical COD is repre-
sented by a broken line, the fungal dry weight by
and the COD by
-67-
-------
In this experiment, the feed rate was held constant at 5 ml/min. in an
18-liter fermentation. Nutrient exhaustion and physiological aging of
the fungi was prevented by removal of part of the fungal mass whenever
it exceeded preset limits. This was in addition to the removal that
constantly occurred in the effluent stream from the fermentor. Control
of mycelial age could be achieved if fungal mass levels were held be-
tween 3.2 and 3.5 grams per liter. The data are shown in Figure 26.
The data for this experiment strongly indicated that COD's below 1000
mg/1 could be achieved with a flow rate of 5 ml/min. in an 18-liter fer-
mentation with control of fungal mass to levels of 3.2 to 3.5 g/1. When
the fungal mass climbed above 4 g/1 at this flow rate, partial fungal
lysis occurred. Partial lysis occurred several times during the past
thirty days and each time this occurred the yeast population rose to a
high level (approximately 109 cells/ml). Restoration and maintenance
of the fungal mass at the critical level (3.2-3.5 g/1) at this flow
rate resulted in reduced COD and massive reduction of the yeast popula-
tion. The minimum COD reached in this experiment was 760 mg/1 which
corresponded to a BOD of 235 mg/1. This was the most successful con-
tinuous fermentation achieved in a single-step digestion on soy wheys.
Although the data plotted in Figure 26 show the results obtained over
a sixteen-day period, the continuous digester was in operation for
thirty days. The near steady state was achieved from the thirteenth
day through the thirtieth day. The first sixteen days are expanded in
Figure 26 to illustrate several features of this fermentation. Initi-
ally, the SOs concentration was 200 mg/1 and was increased to 570 and
then to 760 mg/1 at the times indicated in Figure 26. There appeared
to be a short growth lag at the 570 and 760 mg SOs/l levels. It can
also be seen that when the fungal mycelium exceeded 4 gm/1, the COD
began to rise. The morphological appearance of the mycelium during the
tenth and eleventh days was one of early sporulation and beginning lysis.
Also, yeasts and bacteria began to appear in larger numbers. When the
fungal mass was reduced at the thirteenth day to approximately 3 g/1, by
removing mycelium, the COD rapidly decreased. The reduction in fungal
mass resulted in rapid appearance of new mycelium in the form of elon-
gated tips from the hyphal cross walls. Spores were germinating, and
elongated germ tubes were emerging. After the thirteenth day, extreme
care was exercised to maintain the fungal mycelium between 3.2 and 3.7
g/1.
Secondary Stage Digester. One further attempt to remove the residual
COD remaining after digestion of the S02 soy whey was also included in
this experiment.
Two approaches were open for experimentation: (1) to isolate and char-
acterize the residual materials and then incorporate microorganisms known
to utilize these materials into a secondary stage fermentor or (2) to
select microorganisms through soil enrichment on soy COD residual mate-
rials. The latter approach was tried first with rather interesting re-
sults. Three fungal isolates grew from soil enriched with the soy resid-
ual COD. The isolates were not identified although they were each iso-
lated in pure culture on Czapek-Dox agar. Soy supernatants collected
-68-
-------
12,000
10,000 -
8000-
o> o>
E E
6000-
. Q a
ON O O
f O O
UJ 5
co Q.
4000
2000
THEORETICAL
FEED RATE: 5 ml/mi n
S02
570 mg/1
I I
S03
760 mg/1
"A-
O-
•A-
-o-
I
A-
•O-
I
12,000
10,000
8000
6000
o>
CO
4000
2000
days
8
DAYS
10
12
14
16
0
Fig. 26. Continuous digestion of SOa soy whey by G^ deliquescens. Fungal mass control of fermentation.
Solid line represents the theoretical COD if no removal was achieved. The fungal mass dry weight is repre-
sented by *-- - •, the secondary stage COD reduction by 0 - 0, and the primary (G. de liqe scen
(G.
TT
e COD
A- BOD's were determined as shown by arrows. The SOa concentrations in mgTT are also indicated by
the appropriate arrows.
-------
after removal of G_. deliquescens and which contained 3000 to 6000 mg
COD/1 were reduced to approximately 500 mg COD/1 after 60 hours. These
fungi isolated from soil were tested in a secondary stage fermentor.
A secondary four-liter digestion flask was set up with an aerator, a
feed line consisting of effluent from the primary S02 soy whey digester,
and an exhaust effluent line. This apparatus is shown in Figure 1,
page 12. The fermentor was fed with effluent from the first fermenta-
tion after removing the fungal mycelium by filtration. The secondary
system was inoculated with fungal isolates from soil obtained where soy
beans had grown for several years. No adjustments of pH, temperature,
flow rate, etc., were used for this system. The effluent COD from the
primary digester was further reduced in this system to 550 mg COD/1 and
a BODs of 125 mg/1. These data are shown in Figure 26 during the third
to seventh days and thirteenth to sixteenth days of operation.
Feed Rate Control. In this experiment, nutrient exhaustion and physio-
logical aging were prevented by increasing the feed rate whenever the
mycelium appeared slightly vacuolated or granular. Thus, the experiment
started at a feed rate of 5 ml/min., and the feed rate was increased at
2 ml/min. increments when the microscopic evidence dictated a change.
The data showed a COD variation of 1100 to 1800 mg/1 during the time the
fungus increased in mass from 1.4 to 8.0 g/1. During the course of this
experiment the feed rate was increased from 5 to 13 ml/min. in a 17-liter
fermentation. Although the COD was maintained at a fairly constant level
between 1000 and 1800 mg/1, several features of the experiment should be
noted and are plotted in Figure 27.
The step-wise increase in S02 concentration during the experiment pro-
duced a noticeable lag in fungal growth at each increased S02 increment.
The fungus recovered after each S02 increase and grew at a rate equal to
that observed prior to the S02 addition^' These points are shown in
Figure 27 as S02 concentrations at the second day, the fifth day, and
at the eleventh day.
A second problem was the excessive foaming which prevented an accurate
evaluation'of the fungal mass and COD after sixteen days. The foam was
found to be of higher COD than the bulk liquor of the fermentor. This
meant that enrichment of the foam tended to reduce effective exposure
time of influent COD. The erratic foaming also caused erratic changes
in the digestor volume and a concentration of the fungal mycelium in the
foam. The foaming condition was corrected with constant feeding of Anti-
foam B at a rate of 0.001 ml/min. Although this experiment was conducted
for only sixteen days free of foaming, rather stable COD reduction was
maintained during this time. At the eighth day when the feed rate was
raised to 13-15 ml/min. the fungal mycelium rapidly washed out as shown
by the decreased fungal mass after this time. The reduced fungal mass
would have resulted in an increased COD if the feed rate had not been
lowered to the 11-13 ml/min. level. Thus, control was maintained via
feed rate shifts, and the COD remained fairly constant as shown. In-
creased COD at the eleventh day was probably due to the increased S02
concentration (760 mg/1) which temporarily resulted in minimal lysis of
-70-
-------
12,000
10,000 -
8000-
o>
6000-
Q
O
O
4000-
2000-
I 1 ^ i 1 1 1 \ 1 1 1 1 I I
THEORETICAL
12,000
- 10,000
- 8000
- 6000
- 4000
- 2000
Fig. 27. Continuous digestion of 80s soy whey by G. deliquescens. Feed rate changes indicated
by arrows pointing 5-7, 9-11, 11-13, 13-15 ml/min were used to control the fermentation. The solid
line indicates the theoretical COD which shows a break where the feed rate was increased to 7 ml/min.
The fungal dry weight is indicated by the • ' • line and the actual COD by the A A line. SP, on
the fungal curve, indicates the area where fungal sporulation occurred.
-------
the mycelium. New growth appeared on the thirteenth day, and the COD de-
creased; however, the feed rate, unfortunately, was lowered to 9-11 ml/
min. at a time when it should have been either maintained at 11 to 13 ml/
min. or raised slightly. The result was a starvation condition for the
fungus. Microscopically, the-mycelium showed some spore formation, and
the COD immediately rose to 3000 mg/1. The experiment was discontinued
at this point. It appeared from this study that the optimal feed rate
required to stabilize the COD between 1500-2000 mg/1 was somewhere be-
tween 9 and 13 ml/min. This was equivalent to a turnover time, or re-
tention time, of 24 to 36 hours.
Chemical Composition
The reduction in certain components of a mixed SOg soy whey following
continuous digestion by G_. deliquescens are shown in Table 18. The
chemical analyses were carried out on aliquots of samples removed from
the continuous fermentation on day 7 in Figure 26.
Table 18
Reduction of the Chemical Components* of SOg Soy Whey by G. deliquescens
1
Test
COD
BODs
Carbohydrate
Protein
Phosphate
Total
Nitrogen
(Kjeldahl)
S02
Sulfates
Chlorides
Fungus
Solids
Ash
2
Raw S02
Soy Whey
mg/1
11,200
8,130
4,700
3,950
122
1,485
700
196
230
—
10,600
2,120
3
Raw HC1
Soy Whey
mg/1
14,480
9,750
7,000
4,000
203
1,524
—
220
2,700
—
14 , 800
3,230
4
Mix
75 S02
25 HC1
Soy Whey
mg/1
12,230
8,537
5,450
3,950
144
1,514
525
208
848
—
11,650
2,397
5
After
Fungal
Digestion
mg/1
Pri.
Stage
808
235
215
420
43
148
20
102
550
3300
2800
1620
Sec.
Stage
648
125
6
7» Reductions
Pri.
Stage
93.4
97.3
97.0
89.4
70.0
90.2
96.0
50.0
35.0
—
76.0
32.0
Sec.
Stage
94.7
98.6
Analyses performed on samples of the effluent were done after filtering
through a single layer of Whatman No. 4 filter paper.
-72-
-------
The values in Columns 2 and 3 (Table 18) are actual analyses of the two
(80s and HC1) raw soy wheys studied in this work. When the continuous
digester effluent was analyzed (day 7), the two raw soy wheys were mix-
ing in the feed tank in the proportion 75:25 as shown in Column 4. Thus,
the chemical levels in Column 4 were mathematical expressions of what was
fed to the digester during Day 7- The results of the G. deliquescens di-
gestion of these chemical components are shown in Column 5 (primary di-
gestor with G. deliquescens) and in Column 5 (secondary digester) for the
soil-enriched digestion of the primary effluent residue.
Most notable were the BOD reductions from an initial level of 8537 mg/1
to 235 mg/1 in the primary digester and to 125 mg/1 in the secondary-
stage digester.
Amino Acid Composition
The amino acid composition of the G. deliquescens mycelium grown on SOs
whey was not significantly different from the same fungus grown on HC1
soy whey (Table 15). However, the amino acid analysis revealed two peaks
not observed in the HC1 soy whey grown G. deliquescens. These were iden-
tified qualitatively as methionine sulfoxide and methionine sulfone.
Rat Feeding
G. deliquescens collected and lyophilized from the continuous soy whey
digestion trials was stored in a freezer. The lyophilized fungal mycel-
ium grown on HC1 soy whey was stored separately from mycelium collected
from the fermentations where SOg soy whey was used as the growth medium.
There was not enough dried mycelium from either growth medium type (HC1
or S02 soy wheys) to carry out separate rat feeding trials. Therefore,
the mycelium grown on both soy wheys were pooled and mixed with a special
Nutritional Biochemical Corporation (NBC) diet which contained starch,
fats, vitamins, and salts equivalent (except for protein) to the NBC
standard weanling rat casein diet. The pooled fungal mycelium, which
contained 46 percent protein, was mixed 50:50 with this special NBC form-
ulation to provide a complete diet which contained 23 percent fungal pro-
tein. The standard diet was prepared in the same manner except that the
23 percent protein source was casein. These were the same diets as
those used for the corn waste T?. viride feeding experiments - except
that (5. deliquescens mycelium replaced the T. viride mycelium.
The whole fungal mycelium was used as the protein source and, based on
the comparative amino acid analyses of the fungal protein and of casein,
certain L-amino acids were added to supplement both proteins as required
for weanling rats. Sulfur amino acids were low in both proteins and L-
methionine was added to both standard (casein) and the test (fungal)
diets. Other L-amino acid (L-methionine, L-serine, L-valine, L-leucine,
L-tyrosine and L-glutamic) which totaled <5 percent of the total amino
acids of the test diet, were added to the fungal protein diet to equate
the amino acid composition to that provided in the casein diet. Eight
rats were placed in separate metabolic cages and fed the standard (casein)
-73-
-------
diet plus 1 percent chromic oxide. When all rats showed green fecal mate-
rial (24 hours), four were placed on the test (fungal) diet. Fecal and
urine samples were collected daily. Weight gain or loss, signs of toxic-
ity, food consumed, etc., were recorded daily for each rat.
Table 19 shows the results of this feeding experiment, where the test
(fungal) diet contained the combined protein of G_. deliquescens grown on
HC1 and S02 soy wheys. The results show that the rats refused to eat the
test diet and lost weight each day. After the third day, three rats (two
shown here) were given glutamate with the fungal diet in the belief that
this might improve palatability of the test diet. The rats on the test
diet with glutamate did eat more of the fungal diet but continued to lose
weight. All test rats died when their bodyweight reached approximately
fifty grams, as shown (Final Rat Weight) in Table 19. Only two of the
four standard diet fed rats and three of the test-diet-fed rats are shown
here. This was done for the sake of brevity - the other data do not de-
viate significantly from these.
Another experiment was set up to test the palatability of this fungal
mycelium. Rats were fed the fungal mycelium as their sole food source.
Thus, two rats received HC1 soy whey grown G. deliquescens, two received
S02 soy whey grown G_. deliquescens, two received aqueous extracted S02
soy grown mycelium and two received 95 percent ethanol-extracted S02 soy
whey grown mycelium. The results of this study are shown in Table 20.
The results are average values for the two rats fed each experimental
fungal diet.
Table 20
Rat Feeding Trial
Fungal Mycelium as Sole Source of Diet
Day
1
2
Total
Fungus Grown on
HC1 Soy Whey
Keed
Consumed
gm
2
3
5
Weight
Gain
gm
-1
4
3
Fungus Grown o'h
S02 Soy Whey
Feed
Consumed
gm
0
0
0
Weight
Gain
gm
-6
-9
-15
Fungus Grown on
S02 Soy Whey
H20 Extracted
Myce 1 ium
Feed
Consumed
em
0.2
0.25
0.45
Weight
Gain
gm
-4
-6
-10
Fungus Grown on
S02 Soy Whey
EtOH Extracted
Mycelium
Feed
Consumed
gm
4
5
9
Weight
Gain
gm
3
5
8
These feeding trials (Tables 19 and 20) showed the mycelium harvested from
SOg-containing soy whey to be impalatable to weanling rats. The impalata-
bility was removed by ethanol extraction but not by water extraction. The
ethanol extract itself proved neither toxic nor impalatable. This was ob-
served when rats were intubated with a fourteen-fold concentrated, ethanol
-74-
-------
Table 19
Rat Feeding Trial Casein vs. G. deliquescens Test Diet
Day
1
2
*3
4
5
6
7
Total
Initial
Rat Wt.
Final
Rat Wt.
Standard Casein Diet
Feed Weight
Consumed Gain
gm gm
9.9 6
11.9 7
12.5 9
12.0 8
11.5 5
12.1 6
13.1 9
83.0 50
80
130
Feed Weight
Consumed Gain
gm gm
10.0 7
11.9 2
13.8 16
12.7 8
13.6 9
13.8 10
14.7 5
90.5 57
80
137
Test Fungal Diet
Feed Weight
Consumed Gain
gm gm
1.4 -7
2.3 -8
*3.1 -1
3.7 -3
3.3 -5
2.1 -5
0.2 -1
16.1 -30
80
50
Feed Weight
Consumed Gain
gm gm
0.6 -7
1.3 -7
*1.9 -2
3.2 -1
4.1 -3
3.0 -4
2.8 -2
16.9 -26
82
56
Feed Weight
Consumed Gain
gm gm
0.6 -5
0 -9
0.9 -3
1.0 -7
0.3 -4
2.8 -28
77
49
Day 3 - two rats on test diet received glutamate as indicated by. stars.
-------
extract. Since evaporation in vacuo was used in preparing the extracts
for feeding, loss of aversive character in the ethanol extraction may be
explained by volatility of a critical component.
A third feeding experiment was started in which HC1 soy whey grown G.
deliquescens was mixed with the NBC special preformulated diet described
in the Methods section and fed to two rats along with two other rats pre-
sented the standard casein diet. This feeding experiment was run in the
same manner as first described for the corn and soy grown fungi. The
purpose of this experiment was to ascertain what, if any, effect the soy
whey (minus SOg) medium had on the fungal mycelium when used as a feed.
Because of the limited quantity of HCl-soy-grown mycelium, only two rats
could be used in the test diet. These rats were fed for only seven days
before the feed was exhausted. A plot of the rat growth is presented in
Figure 28. After an initial lag, the test animals began to gain weight
on this diet. The growth rates, as determined by the slopes of the
curves, were 7.0 for the standard rats and 5.0 for the test animals.
None of the test rats died nor did they show any toxic symptoms during
this one-week feeding trial. One month later the test rats were healthy
and equal, in weight, to the rats fed only the standard casein diet.
Consumption of mycelium grown on HC1 soy whey was initially low and may
be indicative of a moderate palatability problem with this material,
also. Limited supplies did not permit really meaningful experiments.
-76-
-------
100
-J
^J
I
Fig. 28. Weanling rat growth rates fed a standard casein diet and a test G_^ deliquescens
fungal diet. Slopes of weight gains are as indicated in figure.
-------
SECTION VI
ECONOMIC ESTIMATES
Only crude estimates of the cost of waste treatment by fungi can be made
from laboratory data alone. Larger-scale trials will be required as a
basis for more accurate estimation. Estimates for costs of application
of fungi to corn processing wastes are summarized in Table 21. The
estimates were based on recovery of 0.5 pound of dried mycelium for
each pound of COD utilized. This is a conservative estimate based on
accumulated experience.
Table 21
Economy of Corn Waste Treatment
Cents per
Ib fungal
Item Amount product
N (NH4)2S04 0.45 Ib 0.67
P04sNaH2P04 0.022 Ib 0.20
H2S04 0.10 Ib 0.14
Aeration 0.28 Ib dissolved oxygen
(1 hp hr = 2 Ib DO) 0.16
(Power cost = 1.5
-------
is meant to cover the cost of the aeration equipment, the lagoon, and
costs of control equipment. This estimate also seems reasonably conser-
vative. The investment has been amortized over 10 years at 8 percent
interest. It was assumed that the COD load is 2500 mg/1 and the equip-
ment is in use 50 days per year.
Labor costs were calculated assuming eight hours of labor a day at $100
cost per day (including overhead) to operate a 2,500,000 gal/day installa-
tion.
Filtering and drying costs represent a gross estimate and are meant to
cover labor, capital equipment, power, and other costs associated with
this operation.
Sales returns assume the product would bring the same price as soy oil
meal with which it compares in protein content and quality.
Estimates for soy waste processing have similarly been attempted (Table
22). Nitrogen and phosphate supplies are probably adequate in the in-
coming feed and so do not need to be added. Aeration requirements are
similar to those of corn per pound of COD removed, but the costs are
lower because the amortization is spread over constant operation in-
stead of over fifty days operation per year. The constant operation
does raise the need for heat in the winter in northern climates. No
attempt has been made to estimate heating costs because it is not known
whether waste heat would be available from processing operations.
A plant handling 3,500,000 gallons of waste per day with a COD load of
8,000 mg/1 has been assumed.
It has been assumed that one pound of dry product is obtained per two
pounds of COD removed.
Table 22
Economy of Soy Waste Treatment
Cents per
pound
Amount product
0.1 Ib 0.14
0.28 Ib 02/lb product
(1 hp hr = 2 Ib DO) 0.16
(Power cost = 1.5
-------
SECTION VII
ACKNOWLEDGMENTS
The authors wish to express their appreciation to several
persons who have taken a most helpful interest in the study.
Judith Grimes provided most able assistance. Dr. Jose Concon
provided invaluable suggestions and collaboration in feeding
studies and selection of analytical procedures. Dr. Wm.
Bridge Cooke provided many helpful suggestions, references,
and general guidance. Dr. William Gray provided us with
fungal cultures, useful suggestions, and enthusiasm.
-81-
-------
SECTION VIII
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-------
15. Novick, A. 1955. Growth of Bacteria. Ann. Rev. Microbiol. 9: 97-
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by Sphaerotilus. Biotech, and Bioeng. 5: 4-16.
18. Postgate, J. R. and J. R. Hunter. 1962. The Survival of Starved
Bacteria. J. Gen. Microbiol. 29: 233-263.
19. Standard Methods for the Examination of Water and Wastewater. 1965.
Oxygen Demand (Chemical). Amer. Pub. Health Assoc. New York, New York.
12th Ed. p. 510-514.
20. Standard Methods for the Examination of Water and "Wastewater. 1965.
Oxygen Demand (Biochemical). Amer. Pub. Health Assoc. New York,
New York. 12th Ed. p. 415-421.
21. Standard Methods for the Examination of Water and Wastewater. 1965.
Determination of chloride by the mercuric nitrate method; nitrate,
by the phenoldisulfonic acid method; and sulfate, by the turbidi-
metric method using HC1 and BaCl. Amer. Pub. Health Assoc. New York,
New York. 2nd Ed. pp. 87-89, 195-198 and 291-293, respectively.
22. Lilly, V. M. and Ho L. Barnett. 1951. Physiology of the Fungi.
McGraw-Hill Book Co. 1st Ed. New York, New York.
23. Cochrane, V. W. 1958. Physiology of the Fungi. John Wiley & Sons,
Inc. New York, New York.
-84-
a U. S. GOVERNMENT PRINTING OFFICE : 1970 O - 403-032
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1
5
Accession Number
2
Subject Field & Group
05D
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Organization
North Star Research and Development Institute
3100 38th Avenue South
Minneapolis, Minnesota 55406
Title
Use of Fungi Imperfecti in Waste Control
10
Authors)
Brooks D. Church
and
Harold A. Nash
16
Project Designation
12060 EHT
21
Note
In conjunction with:
The Green Giant Company
General Mills, Inc.
Central Soya Company
Ralston-Purina Company
22
Citation
23
Descriptors (Starred First)
Fungi Imperfecti
Corn waste
Soy whey
BOD removal
Fungus feed
Economic costs
25
Identifiers (Starred First)
27
Abstract
Species of Fungi Imperfecti were screened for those candidates best able to convert
soluble and suspended organic material from corn and soy food processing waste streams to
mycelial protein. Optimal growth conditions of the selected fungal strains included pH of
3.2 to 3.5 and a temperature of 30°C. Oxygen requirements were relatively low (1 Ib Os/6
or 7 Ib COD removed). Nitrogen and phosphate additions were required for the corn diges-
tion system, and additions of HsS04 were used to adjust the pH. These studies were done
in batch and continuous culture systems using nonsterile corn and soy waste. Corn waste
was reduced from an initial BOD of 1600 mg/1 to 25 mg/1 in 24 hrs and soy waste from 6200
mg/1 to 125 mg/1 in 36 hrs. Studies of rapid fungal digestion of soy whey containing 700
mg/1 of SOs resulted in selections of 2 fungal genera which removed SOg from the waste
medium. Mycelial yields were 50-60 g/100 g of COD utilized. Protein content of the fun-
gal mycelium was 45 percent. Feeding trials in weanling rats gave a growth response equal
to that seen with a standard casein rat diet. Digestibility was 90 percent and no toxicity
was observed in a three-week trial. Economic estimates based on the experimental results
showed the fungal product to be comparable in cost to soy oil meal.
This report was submitted in fulfillment of Grant No. 12060 EHT between the Federal
Water Pollution Control Administration and North Star Research and Development Institute.
Abstractor
Brooks D. Church
Institution
North Star Research and Development Institute
WR:I02 (REV. JULY 1969)
WRSIC
SEND TO: WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON. D. C. 20240
* SPO: 1969-359-339
-------
BIBLIOGRAPHIC:
North Star Research and Development Institute,
Use of Fungi Imperfect! in Waste Control, Final
Report FWQA Grant No. 12060EHT07/70.
ABSTRACT
Species of Fungi Imperfectl were screened for
those candidates best able to convert soluble and
suspended organic material from corn and soy food
processing waste streams to mycelial protein.
Optimal growth conditions of the selected fungal
strains included pH of 3.2 to 3.5 and a tempera-
ture of 30° C. Oxygen requirements were relatively
low (1 Ib 02/6 or 7 Ib COD removed). Nitrogen and
phosphate additions were required for the corn
digestion system, and additions of l^SO^ were used
to adjust Che pH. These studies were done In batch
and continuous culture systems using nonsterilc
corn and soy waste. Corn waste was reduced from an
initial BOD of 1600 mg/1 to 25 mg/1 in 24 hrs. and
soy waste from 6200 mg/1 to 125 mg/1 in 36 hrs.
ACCESSION NO.
KEY WORDS:
Fungi Imperfect!
Corn Waste
Soy Whey
BOD Removal
Fungus Feed
Economic Costs
BIBLIOGRAPHIC:
North Star Research and Development Institute,
Use of Fungi Imperfect! in Waste Control, Final
Report FWQA Grant No. 12060EHT07/70.
ABSTRACT
Species of Fungi Imperfect! were screened for
Chose candidates best able to convert soluble and
suspended organic material from corn and soy food
processing waste streams to mycelial protein.
Optimal growth conditions of the selected fungal
strains included pH of 3.2 to 3.5 and a tempera-
ture of 30° C. Oxygen requirements were relatively
low (1 Ib ©2/6 or 7 Ib COD removed). Nitrogen and
phosphate additions were required for the corn
digestion system, and additions of t^SO^ were used
to adjust the pH. These studies were done in batch
and continuous culture systems using nonsterile
corn and soy waste. Corn waste was reduced from an
initial BOD of 1600 mg/1 to 25 mg/1 in 24 hrs. and
soy waste from 6200 mg/1 to 125 mg/1 in 36 hrs.
ACCESSION NO.
KEYWORDS:
Fungi Imperfect!
Corn Waste
Soy Whey
BOD Removal
Fungus Feed
Economic Costs
BIBLIOGRAPHIC:
North Star Research and Development Institute,
Use of Fungi Imperfect! in Waste Control, Final
Report FWQA Grant No. 12060EHT07/70.
ABSTRACT
Species of Fungi Imperfectl were screened for
those candidates best able to convert soluble and
suspended organic material from corn and soy food
processing waste streams to mycelial protein.
Optimal growth conditions of the selected fungal
strains included pH of 3.2 to 3.5 and a tempera-
ture of 30° C. Oxygen requirements were relatively
low (1 Ib 02/6 or 7 Ib COD removed). Nitrogen and
phosphate additions were required for the corn
digestion system, and additions of l^SO^ were used
to adjust the pH. These studies were done in batch
and continuous culture systems using nonsterile
corn and soy waste. Corn waste was reduced from an
initial BOD of 1600 mg/1 to 25 mg/1 in 24 hrs. and
soy waste from 6200 mg/1 to 125 mg/1 in 36 hrs.
ACCESSION NO.
KEY WORDS:
Fungi Imperfect:
Corn Waste
Soy Whey
BOD Removal
Fungus Feed
Economic Costs
-------
Studies of rapid fungal digestion of soy whey contain-
ing 700'mg/1 of S02 resulted In selections of 2 fungal
genera which removed SO2 from the waste medium.
Mycelial yields were 50-60 g/100 g of COD utilized.
Protein content of the fungal mycelium was 45 percent.
Feeding trials in weanling rats gave a growth response
equal to that seen with a standard casein rat diet.
Digestibility was 90 percent and no toxicity was
observed in a three-week trial. Economic estimates
based on the experimental results showed the fungal
product to be comparable in cost to soy oil meal.
This report was submitted in fulfillment of Grant
No. 12060 EHT between the Federal Water Pollution
Control Administration and North Star Research and
Development Institute.
Studies of rapid fungal digestion of soy whey contain-'
ing 700 mg/1 of 502 resulted in selections of 2 fungal
genera which removed SC>2 from the waste medium.
Mycelial yields were 50-60 g/100 g of COD utilized.
Protein content of the fungal mycelium was 45 percent.
Feeding trials in weanling rats gave a growth response
equal to that seen with a standard casein rat diet.
Digestibility was 90 percent and no toxicity was
observed in a three-week trial. Economic estimates
based on the experimental results showed the fungal
product to be comparable in cost to soy oil meal.
This report was submitted in fulfillment of Grant
No. 12060 EHT between the Federal Water Pollution
Control Administration and North Star Research and
Development Institute.
Studies of rapid fungal digestion of soy whey contain-
ing 700 mg/1 of S02 resulted in selections of 2 fungal
genera which removed S02 from the waste medium.
Mycelial yields were 50-60 g/100 g of COD utilized.
Protein content of the fungal mycelium was 45 percent.
Feeding trials in weanling rats gave a growth response
equal to that seen with a standard casein rat diet.
Digestibility was 90 percent and no toxicity was
observed in a three-week trial. Economic estimates
based on the experimental results showed the fungal
product to be comparable in cost to soy oil meal.
This report was submitted in fulfillment of Grant
No. 12060 EHT between the Federal Water Pollution
Control Administration and North Star Research and
Development Institute.
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