DEGRADATION OF WASTE PAPER
TO PROTEIN
Research in Microbial Fermentations
US. ENVIRONMENTAL PROTECTION AGENCY
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This report has been reviewed by the U.S. Environmental Protection
Agency and approved for publication. Approval does not signify that
the contents necessarily reflect the views and policies of the U.S.
Environmental Protection Agency, nor does mention of commercial
products constitute endorsement or recommendation for use by the
U.S. Government.
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DEGRADATION OF WASTE PAPER TO PROTEIN
Research in Microbial Fermentations
This final open*-file report (SW-16rg
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An environmental protection publication
in the solid waste management series (SW-16rg.of)
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CONTENTS
"'" Page
Background Material 1
Introduction 2
Methods 2
Shake Flask Studies 4
Stirred-Jar Fermentoir Studies 12
Discussion 15
Tables I through XXXI 17
Figures 1 through 19 77
References 86
Publications Supported by Grant 88
Staffing 88
Append ix 89
Table I, Culture Media Used for Isplation pf 89
Hydrocarbon-Oxidizing Organisms and Some Cellulose Utilizers
Table II, Culture Media Used Primarily for Cellulolytic 91
Microorganisms
Original Biuret Protein Method 94
Revised Biuret Protein Method 97
Calculation of Corrected Protein and Cellulose Values 99
A. Shake Flasks
B. Stirred-Jar Fermentors
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ABSTRACT
The early stages of this work concentrated on efforts to isolate
pure cultures which would utilize both cellulose and hydrocarbons.
From more than 70 different inocula of soil, compost, sewage sludge
and forest litter, 367 pure cultures were isolated on n-hexadecane
media; 28 of these were found to be capable of attacking sodium
carboxymethyl cellulose and lowering its viscosity. Only one proved
to be capable of attacking purified cellulose, ball-milled newspaper,
or newsprint. This culture is a fungus which grows well up to 45 C,
identified as Aspergillus fumigatus.
More than 300 enrichment cultures were also developed on cellu-
losic substrates (purified cellulose, ball-milled newsprint, without
ink, or ball-milled newspaper) from similar inocula. These were
tested in shake flask fermentations for ability to produce protein
and utilize the cellulosic substrates listed above. Only 10 enrich-
ment cultures yielded greater than 0.2 mg of protein per ml in 5 days.
These also utilized appreciable amounts of cellulose. From the active
enrichment cultures, 5 cellulolytic pure cultures were isolated. All
of these were fungi. All gave considerably higher protein yields and
cellulose consumption under shake flask conditions than the known
cellulolytic organisms Trichoderma viride or Cellulomonas sp. These
organisms were studied in shake flask fermentors in an effort to
achieve optimum rates of cellulose utilization and protein synthesis.
The final identification of the five fungus cultures was made by
Dr. Richard N. Kinsley, Jr., for which we express our gratitude and
appreciation. Two of the cultures were Myrothecium verrucaria, two
were Aspergillus fumigatus and one was Trichoderma lignorum.
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Myrothecium verrucaria gave the highest rates of protein synthesis
of any of the fungi studied in shake flask studies; therefore, follow-up
studies using stirred-jar fermentors concentrated on this organism
(culture P9). Conditions were optimized during 12 runs in 14-liter stirred-
jar fermentors. Protein yield on ball-milled newspaper increased with
substrate concentration up to the maximum used. It was shown that a very
simple medium containing dibasic ammonium phosphate, 1%, urea, 0.03%, and
yeast autolysate, 0.1%, was optimal for growth and protein synthesis on
ball-milled newspaper, 4 g per 100 ml of medium. Under these conditions
(run 11) the maximum rate of cellulose consumption was 5.4 g/l/day, and
the rate of protein synthesis was 0.3 g/l/day. The maximum yield of
protein obtained was 1.42 g/1 by the highly specific modified biuret
method, or 3.3 g/1 by usual method of multiplying total organic (Kjeldahl)
nitrogen by 6.25. The amount of cellulose consumed in this experiment
was 12.7 g/1 from an original 20.4 g/1 contained in 40 g/1 of ball-milled
newspaper.
The final product was recovered by evaporation of the culture fluid.
Chemical analysis indicates that it may be a nutritious animal feed, but
this must be proved by the construction of the pilot plant to manufacture
sufficient matetial for extensive animal feeding studies to evaluate
toxicity and nutrient value.
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Background Information
Cellulose is perhaps the most abundant of all organic materials in
nature, as it is the major structural material of most plants. It sometimes
occurs in almost pure state, as in cotton fibers, but more often in association
with other compounds. Wood, for example, is a complex of cellulose (40 to 80%),
lignin (20 to 307.) , hemicelluloses and xylosans (10 to 30%) .^ The ]ignocellulose
complex of softwood is attacked only with great difficulty by cellulolytic enzymes.
Thus, paper made from "mechanical pulp," such as newspaper which is simply
ground-up softwood, is relatively resistant to degradation by microorganisms.
Paper made from chemical pulp, is much more readily attacked, as the hot sulfurous
acid or alkaline sulfates used in the chemical treatment have partially delign-
ified the wood pulp. Accordingly, we have chosen to carry out most of our work
on newspaper, since any other kind of paper will probably be more easily degraded.
Reese, Siu and Levinson^O showed that many fungi and bacteria are able
to hydrolyze modified cellulose, such as sodium carboxymethyl cellulose, but few
are able to attack native cellulose such as cotton fibers. From these and other
data they developed a general theory of the dual nature of cellulase enzyme
systems. According to this theory, true cellulolytic organisms, those capable
of attacking native cellulose, produce two or more enzymes. The first, called
GI, attacks native cellulose to break up the aggregates and produce linear
chains of anhydroglucose units. These chains are then attacked by a second
enzyme C , a P 1,4-glucanase, which hydrolyzes them to the disaccharide cellobiose.
Cellobiose may then be assimilated directly into the cell, or may be converted
to glucose by P-glucosidase before assimilation. Mandels and Reese^l have pre-
sented the following schematic description of their theory:
cellulose "* reactive ~* cellobiose "* glucose
cellulose
C, .,. C &-glucosidase
1 (linear x ,
I I
unknown chains) hydrolytic hydrolytic
mechanism
These authors also found that woody materials, such as newspaper, were
relatively resistant to cellulases unless they were thoroughly ground by ball-
milling. They also succeeded in separating the C. and C enzymes from Trichoderma
viride filtrates on columns of DEAE dextran. Since several other authors had
questioned the C,, C theory of the dual nature of cellulase enzyme systems,
this represented an important advance. The separation of C. and C activity,
and the demonstration that the combination of the two separated enzymes, C,
and C , was necessary to produce soluble sugars from cotton was also achieved
by Wood using filtrates of Trichoderma koningii.
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Degradation of Waste
Paper to Protein
Introduction
i
In April, 1968, Dr. J. D. Douros prepared a detailed progress report
covering the progress of the project up to that time; on December 30, 1968
and February 28, 1969 other progress reports were submitted by the writer.
Since all those who read this final report may not have seen the earlier re-
ports, they shall be summarized here.
The primary emphasis in the earlier stages was placed on attempts to
discover microorganisms which would utilize both cellulose and hydrocarbons.
Most of the cultures were isolated on hydrocarbon media, and their ability
to utilize cellulose was not evaluated until later. Many other cultures were
isolated on media containing both cellulose and hydrocarbons. In the earlier
work, up to August 1968, 60 different samples of soil and decaying wood and
vegetation were obtained from different areas, some near paper mills, saw mills,
petroleum seeps, compost piles, and forest litters. Also a lyophilized microbial
preparation sold by Reliance Chemical Co. for the degradation of cellulosic
wastes was evaluated, as well as sewage sludge samples.
Methods
The hydrocarbon-oxidizing organisms (337 cultures) were isolated from
the inocula of soil, compost, sludge, etc. by both liquid enrichment in one of
the seven media listed in- Table I of the Appendix supplemented with 1% of
n-hexadecane, or by the sprinkle plate technique, wherein the inoculum is
sprinkled over the surface of the hydrocarbon medium solidified with 270 agar.
Also more than 300 enrichment cultures were developed on many different media
contained in shake flasks, using cellulose, newsprint (no ink) or newspaper
as the major or sole energy source. The enrichment cultures were streaked
out on agar plates containing the isolation substrate, in the case of hydro-
carbons, or regenerated cellulose agar for cellulose or paper-utilizing
cultures, for pure culture isolation. The streaking and isolation step was
repeated, and a Gram stain was made. If visually pure, a fermentation was
carried out for 24 hours under shake flask conditions resembling the isolation
treatment. At 24 hours, a Gram stain was again done, and visually checked for
purity. If pure, technicians prepared a permanent slide and froze three tubes
of the original fermentation broth, and inoculated litmus milk for a second
culture to deep freeze. Cultures for transfer purposes were grown on mineral
salts agar + the isolation substrate. Nutrient agar slants for fermentation
inocula were inoculated from the mineral salts agar slants in order not to
attenuate the culture.
The earlier work was impeded by the lack of a specific analytical method
for cellulose, so that it was not possible, in most cases, to determine whether
cellulose was being consumed, or whether protein was being produced from other
nutrients in the culture medium, such as peptone, yeast extract or hydrocarbons.
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During the period from August to December 1968, accurate analytical methods
were developed for total protein (a modification of the biuret method)3
and for cellulose. The latter is a semi-micro modification of the classical
gravimetric method of Crampton and Maynard (1938)^ in which lignin, hemicellu-
loses and other carbohydrates are extracted with acetic acid-nitric acid
reagent. The purified cellulose which remains is dissolved in 67% H-SO, ,
and determined by the anthrone reagent. The method has recently been
published6.
The preliminary screening of cultures for cellulolytic ability was
carried out by the usual methods: (1) Inoculation into Cellvibrio medium
containing a strip of Whatman No. 1 filter paper. Cellulolytic activity was
indicated by digestion of the paper7. (2) Streaking on plates of regenerated
cellulose agar: Cellulolytic activity was indicated by the formation of clear
zones surrounding the colonies**. (3) Inoculation into CMS medium: Cellulolytic
activity was indicated by liquifaction of the very viscous CMC Medium'.
(4) Assaying the culture for cellulase activity on sodium carboxymethyl cellulose
(CMC, Hercules Co., type 7 MF) by a filter paper disc diffusion assay using
£. coli or P. aeruginosa as assay organisms to detect the glucose produced by
action of the original culture on cellulose or CMC. This assay procedure was
described in detail in Dr. Douros's last progress report. Since it has not
yielded reliable predictions of good celluloiytic activity, it has since been
abandoned in favor of the more accurate biuret and cellulose analyses.
All cultures giving any evidence of cellulolytic activity by any of
these procedures were then studied in shake-flask fermentations. Most of them
were grown on three different cellulosic substrates, and in two or three
different culture media. The cellulosic products employed were purified cellu-
lose , Nutritional Biochemicals Co., ball-milled newspaper, and ball-milled
newsprint (no ink) obtained from the Denver Post. The composition of the culture
media employed are given in the appendix to this report. In the earlier part
of the study, the final fermentation liquors were homogenized and analyzed both
for protein by the biuret method and for cellulose consumed by the method des-
cribed earlier. More recently, since it was found that cultures yielding more
than 0.2 mg/ml of protein (on non-hydrocarbon media) always utilized some
cellulose, the protein analysis alone has been employed as a screening device
for the selection of promising cultures to follow up. The general method
employed in the shake flask experiments is described below:
A one-gram portion of the air dried cellulosic substrate was weighed
into a 500 ml baffled shake flask, and 100 ml of the desired liquid medium was
added. The flasks were plugged with polystyrene foam plugs, and were autoclaved
for 20 minutes at 20 psig. The flask was inoculated from a source culture
using at least 10^ cells in the inoculum, and flasks were incubated for 2 days
on a shaker-incubator at the selected temperature. One ml of this inoculum
culture was then employed to inoculate a second flask of the same culture medium.
The cultures were incubated for 5 days on a rotary shaker incubator at 350
revolutions per minute. Analyses carried out on each culture included cell
count, either by plating or direct microscopic examination, pH, and total protein
by biuret. Some cultures were also assayed for cellulase and some were analyzed
for the amount of cellulose consumed as described. Where cultures did not appear
homogenous, they were treated on a Virtis homogenizer before analysis. The
analytical results were corrected for values determined on identical sterile
control flasks. Biuret analysis of sterile controls gave small "protein" values
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due to colored material in the paper substrate, or in the case of Reese's medium,
to the proteose peptone supplement. These values were subtracted from the values
given by the cultures to obtain "corrected" values for protein. The "percent of .
cellulose utilized" figure was calculated by subtracting the amount of cellulose
found in the culture from that in the sterile control.
The data on all enrichment cultures which gave more than 0.2 mg/ml
protein yield are summarized in Table I. These represent the best-yielding
cultures out of many hundreds. The data on the pure culture fermentations
yielding more than 0.2 mg/ml of protein are given in Table II. The sources
and a brief description of the active cultures are given in Table III.
A study of Tables I, II and III reveals that organisms giving a yield
of 0.2 mg/ml or more of protein on purified cellulose or paper in 5 days are
not common. Out of the 367 hydrocarbon-utilizing cultures, only one (C264)
utilized the cellulosic substrate appreciably, although 21 of these cultures
did utilize sodium carboxymethyl cellulose, and 28 of them reduced the viscosity
of CMC in the presence of n-hexadecane. Other conclusions from these experiments
are: Only 10 promising enrichment cultures were found out of more than 500
tested. From these and other inocula, a total of 5 promising pure cultures
were isolated, all fungi. Three of the pure cultures did well on all 3 cellulosic
substrates (cultures C264, 353x85-1, & 353S99-B). Cultures P-9 and P-10, showed
preferences among the substrates. Both gave better yields on newsprint and
newspaper than on purified cellulose. These pure cultures did as well as
the enrichment cultures from which they were isolated in decomposing cellulose.
The five pure cultures of cellulolytic fungi described in Table III
were inoculated on plates of Sabouraud's agar and Czapek's agar, and incubated
for 30 days at room temperature. Macroscopic and microscopic examinations
were made at intervals in an effort to identify the organisms. The character-
istics of the cultures indicated that we we're dealing with 3 species of fungi;
we were unable to identify cultures P9 and P10, culture 353 x 99B was tentatively
identified as Trichoderma lignorum, and cultures 353 x 85-1 and C 264 were
tentatively identified as Aspergillus fumigatus. These cultures were turned
over to Dr. Richard N. Kinsley, Jr., Associate Professor of Microbiology at
East Tennessee State University, who provided the final identifications given
in Table III, for which we express our gratitude and appreciation.
Unfortunately Aspergillus fumigatus is a known pathogen, being the
cause of Aspergillosis, a pneumonitis of birds, which also occasionally infects
humans^". Therefore cultivation on a larger scale was not believed to be
advisable.
Optimization of Growth
Rate of Pure Fungus Cultures
The five pure cultures described above were grown under different
conditions of time, temperature, and medium composition in 500 ml shake
flasks containing- 1 g of ball-milled newsprint and 100 ml of medium with
shaking at 350 RPM. Direct microscopic examinations, pH measurements,
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protein analyses by the biuret method, and cellulose analyses were made
at intervals of 2, 5 and 9 days. The data are given in Table IV.
The conclusions drawn from a study of Table IV are summarized below:
1. P-9 and P-10 grow well at 30 C and 37 C. Protein synthesis is
most rapid at 30 C, but cellulose utilization is greater at 37 C. These
cultures grow very poorly or not at all at 45 C.
2. 353 x 85-1 and C264 grow well at all three temperatures. Protein
synthesis is more rapid at 37 C and 45 C than at 30 C. Cellulose utilization
is also greater at 37 C and 45 C.
3. 353 x 99B grows, synthesizes protein, and utilizes cellulose well
at 30 C, very poorly at 37 C and not at all at 45 C. Culture No. 72 appears
to be an exception to this, but it is believed to have been contaminated with
353 x 85-1 or C-264.
4. P-9, P-10 and 353 x 99B synthesize slightly larger amounts of protein
in 5 or 9 days than 353 x 85-1 or C-264. The percentage of cellulose utilized
was not significantly different among the different cultures for those flasks
giving high protein yields.
5. All actively growing cultures produce acid, as shown by decreasing
pH, but P-9 and P-10 produce slightly more than the other cultures.
6. All of the cultures are very actively cellulolytic, and utilize
more than 80% of the cellulose in 9 days under optimum conditions.
7. TV medium is superior to SM for P-9 and P-10, the two media are
equal for 353 x 85-1 and C-264, and SM is slightly superior for 353 x 99B
for protein synthesis. However, TV is slightly superior for cellulose
utilization by 353 x 99B.
Two remaining aspects of optimization, paper concertration and medium
simplification, were then investigated in two additional experiments.
All other factors being equal, the rate of biosynthesis of cell protein
should increase with increasing substrate concentration. A preliminary experi-
ment was made with ball-milled newsprint (no ink) and ball-milled newspaper.
With both materials, it was found possible to disperse up to 30 g of paper
in 100 ml of aqueous medium without making the system too viscous for agitation
on a shaker. On the other hand, when newspaper was pulped in a Waring blendor
with aqueous medium, a maximum of 2 g of paper could be dispersed in 100 ml
of medium before the system became too viscous to shake. Microscopic examina-
tion revealed the reason. The blended newspaper consisted mostly of long fibers,
whereas the fibers were relatively short in the ball-milled paper.
A preliminary experiment was set up using cultures P9, PlO, and P-26
to evaluate comparative growth on different concentrations of ball-milled
newsprint, newspaper, purified cellulose and blended newspaper in Reese's
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medium. The evaluation of growth was carried out by direct phase contrast
microscopic examination of samples from the shake flasks after 2 days' and
5 days' growth.
The data, summarized in Table V, indicate that fair to good growth
may be obtained on any concentration of any of these cellulosic substrates.
There appears to be some degree of growth inhibition at the highest con-
centration of paper, but since the effect is slight, it could probably be
overcome by adaptation of the inoculum cultures.
Another objective of these experiments is to develop the lowest cost
process possible. This necessitates the use of simple treatment methods and
cheap reagents. Chemicals for media should be low cost fertilizer grade,
and ordinary tap water should be used in place of distilled water. Some
prices, selected from the Oil Paint and Drug Reporter are given below:
$/Ton $/Ton of N
1. (NH4)2S02 (fert. grade, 21% N) 33 157
2. NH4N03(33.5%N) 45 134
3. (NH4)2HP04 (tech. grade, 15% N & 45% P^ 84 466
4. Urea (fert. grade, 46% N) 92 199
5. Anhydrous ammonia (fert. gradej 82.57= N 50 61
6. Phosphoric acid (IUPO, , fert. grade)
52-54% available phosphoric acid , 53
7. (NH,)?HPO,(fert. grade, made by reacting
5 (anhydrous ammonia) and 6(phosphoric acid) 52 348
These chemicals are available in car-load lots at prices far below individually
packaged chemicals. Thus it would probably be advantageous to use these crude
materials wherever possible, blending them to obtain as many of the essential
mineral nutrients as possible. Those mineral nutrients not supplied in the
mixture would probably be supplied in adequate amounts from the paper and
tap water used.
The major nutrients not supplied in adequate quantities by the waste
paper and tap water are nitrogen and phosphorous. Nitrogen could be obtained
from either ammonia, urea, or any of the ammonium salts listed. Phosphate
could be obtained from phosphoric acid, or from ammonium phosphate. It is
clear from the table that nitrogen could be obtained most cheaply from
fertilizer grade anhydrous ammonia, and phosphate from fertilizer grade
phosphoric acid.
In order to evaluate these factors, culture media were prepared using
1% fertilizer grade (NH^SO, and 1% reagent grade (NH^-HPCX (since fertilizer
grade was not available at the time), supplemented with Z% ball-milled newspaper.
Flasks containing the same substrates in Srinivasan's medium were included for
comparison. Shake flasks were set up as previously described, inoculated with
5 day old cultures of the same 3 test fungi, and incubated at 30°C. Cultures
were harvested at intervals of 2, 5 and 9 days, and analyzed for protein; pH
was also measured as described earlier.
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The data are presented in Table VI. The conclusions derived from
these data are that growth of all 3 fungi is slightly more rapid in
Srinivasan's medium during the early growth phase, as shown by a higher pro-
tein yield in 2 days, but yields in 5 and 9 days were far superior on the
ammonium phosphate medium (G). Yields on the ammonium sulfate medium (F) were
approximately equal to those on Srinivasan's medium. Considering that
Srinivasan's medium contains 6 different salts, and yeast extract, the superiority
of medium G, containing only a single salt in tap water is remarkable.
As a follow up on the last experiment with media F and G, prepared
with tap water and ammonium sulfate and tap water and ammonium phosphate,
an experiment was set up with medium H, containing equal parts of the two
salts. The other conditions were the same except that the inoculum cultures
were taken from the preceding experiment in medium G, and were 16 days old,
liaving been stored in the refrigerator for a week after the 9 days' incubation.
The data are summarized in Table VII. The data indicate lower rates
of protein synthesis, as well as lower final protein yields, than for the
preceding experiment. Medium H appears to be approximately as good as
medium F, but inferior to medium G. The slower rate of protein synthesis
in this experiment may have been caused by the difference in inoculum, that
is the longer incubation time or the lack of an organic nutritional supplement
in the medium. The inoculum for the preceding experiment was grown in
Srinivasan's medium, which is supplemented with yeast extract, whereas
medium G contains no such supplement. Microscopic examinations revealed
very heavy conidiospore formation by Myrothecium verrucaria in Srinivasan's
medium and Reese's medium, but little or no conidiospore formation in
medium G.
Cellulomonas
An experiment in which a culture of Cellulomonas sp., obtained from
Dr. V. R. Srinivasan of L.S.U. , was compared with 4 of our bacterial isolates
closely resembling Srinivasan's culture in cultural characteristics
morphology, and certain biochemical reactions, is summarized in Tables VIII
and IX. Table VIII indicates that all 5 cultures had similar morphology,
and all grew well in both media and digested filter paper and CMC at similar
rates. All were small Gram-variable rods, non-motile, methyl red negative,
Voges-Proskauer negative, indole negative and reduced nitrate to nitrite.
The oxidation-fermentation test showed a negative reaction on glucose, but
slight fermentation by some on starch, cellobiose, sucrose or lactose.
Table IX shows that all grew in the fermentation media on paper and purified
cellulose, but produced very low protein yields in 5 days. All of the isolates
are probably Cellulomonas, although not necessarily the same species. Since
they did not produce good protein yields on newspaper, no further studies
were made on these bacteria.
A culture of an actively cellulolytic strain of Trichoderma viride
(QM No. 6a) was obtained from Dr. T. K. Ghose of the U.S. Army Laboratories,
Natick, Mass., and grown on Reese's medium with 1% newsprint and Srinivasan's
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medium with 17» newsprint. The data, presented in Table X indicate extensive
utilization of cellulose, but poor protein yield at 37°C , but good protein
yield at 30 C. The yields on this fermentation, carried out under the same
conditions as those of Table IV were closely comparable with those obtained
with P9 and P10 (Myrothecium verrucaria) and P26 (Trichoderma lignorum) .
Chemical Treatment of Paper
Newspaper (Wall Street Journal) was pulped in a Waring Blendor with
500 ml of distilled water for each 10 g of paper. The paper was filtered
out and dried in an oven at 105 C. After cooling at ambient conditions,
each portion was weighed and treated with 1 M solutions of the test reagents,
NaOH, H2S04, H3P04 and NaHSO,. One series was stirred with the reagent
at room temperature for 8 hours, and allowed to soak for 4 days.. Samples
were then filtered out, washed with tap water until washings were neutral,
and dried and weighed as before. Another series was heated in a, steam
autoclave at 19 psi for one hour, allowed to soak for four days, filtered
out, washed and weighed. The data are given in Table XI.
The chemically treated paper samples were then dispersed in medium H
and fermented in the usual manner along with other samples containing ball-
milled paper. The inoculum was taken from the experiment described in
Table XI, and was grown in medium H for 5 days. The data are given in
Table XII. Protein yields were disappointingly low for all samples, even
those grown on ball-milled paper, probably reflecting a poor sta-te of the
inoculum. Chemically treated paper gave good growth, as indicated by
microscopic examinations, but protein yields were below those from ball-
milled paper in nearly all cases. None of the chemically treated papers
appeared to give protein yields superior to paper treated with plain water.
Effect of Growth Medium on Inoculum
An experiment was then set up to determine whether protein production
is superior from flasks inoculated from a vegetative seed grown on Reese's
medium or on medium G. Microscopic examination of the inoculum cultures
after 2 days' incubation revealed much heavier growth in Reese's medium,
and the presence of a large number of conidiospores in Reese's medium
only. The experiment also compared medium G with medium H. The 5-day
protein yields (see Table XIII) were far superior from harvest flasks
inoculated with cultures grown in Reese's medium. Cultures P9 and PlO
gave essentially equal protein yields on medium G and H, but P26 gave a
better yield on medium G. The protein yields from P9 and PlO were superior
to those from P26.
Cellulose Synthesis by Fungi
An experiment was then set up to determine the amount of cellulose
synthesized by fungi P9, PlO, P26 and C264 grown on 10% glucose in Reese's
medium. The data, given in Table XIV, indicate that P9 and PlO contain no
cellulose, while P26 and C264 contain considerable cellulose. P9 and PlO
synthesized more protein then P26 or C264.
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Ball-milled vs. Dry-chopped Paper as a Substrate
An experiment was carried out to compare protein yields from P9
and C264 grown on Medium H with 2% newspaper chopped dry in a Waring Blender
vs. 27» ball-milled newspaper. The data, summarized in Table XV indicate
considerably higher 5-day yields from ball-milled newspaper.
Effect of pH, Buffers and Trace Elements
An experiment was set up sto determine the effect of pH, trace
elements and buffering by means of CaCO~ on cellulose utilization by fungi
growing on newspaper in Medium G. The media contained 0.005% bromcresol
green in order to permit visual pH determination. The pH values of flasks
21-30 were adjusted on the third, fourth and fifth days of incubation by
dropwise addition of sterile 0.1 N NaOH. Biuret analyses of protein could
not be done because of interference from the indicator dye, but cellulose
utilization was determined on all flasks. The data, given in Table XVI,
indicate slightly higher cellulose utilization in the absence of the
trace-elements solution than in its presence, and higher cellulose utiliza-
tion in those cultures maintained at pH 5.6 to 5.8 by daily titration than
in those buffered with CaCCX, (terminal pH 4.9 to 5.3) or those allowed to
seek their own pH level (initial pH 4.8 to 5.3, terminal pH 3.1 to 3.9).
Effect of Trace Elements Proteose Peptone and Urea
An experiment was performed to determine the effect of added trace
elements, urea and proteose peptone on protein yield and cellulose utilization
by fungi growing on newspaper in medium G. The interactions of these variables
were studied in both inoculum and harvest cultures. The 5-day harvest data
(see Table XVTl) on protein yields and cellulose utilization indicated
no consistent effect of trace elements, a slight stimulatory effect on protein
synthesis by peptone, and definite stimulatory effect from urea on both
protein yield and cellulose utilization. The flasks containing urea also
had higher pH values (from 4.4 to 5.9 vs. 3.4 to 4.2 in flasks without urea).
This, taken with the results of previous experiments, suggests that urea may
act merely by keeping the pH in the optimum range. Assuming that the fungi
produce urease, the hydrolysis of one mole of urea to 2 moles of ammonia
and one mole of carbon dioxide would decidedly increase pH.
A follow-up on this experiment explored the interaction of these
variables in other combinations, and compared 2-day growth and protein
yield from vegetative seed cultures grown on Reese's medium and medium G
supplemented with urea, urea and trace elements, and urea, trace elements
and protopeptone. Data are given in Table XVIII. Growth and protein yield
in two days was by far the best in Reese's medium. Again, urea appeared
to increase yield markedly, while the trace elements and protopeptone had
little effect.
Earlier experiments showed that Reese's medium gives more rapid
growth than medium G, even when medium G was supplemented with trace elements,
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urea and protopeptone , but medium G gave higher protein yields after
five days. Microscopic examination showed that Reese's medium caused
cultures P9 and PlO to produce very large numbers of conidiospores as well
as some mycelium, whereas only the mycelial form was produced in medium G.
Correlated with this, Reese's medium gave far better results for the
vegetative seed than medium G. An experiment was therefore initiated to
study the effect of substituting other materials for protopeptone in Reese's
medium and Medium G. The data, given in Table XIX, show that casatnino acids,
is as good as protopeptone in promoting growth and protein synthesis in
Reese's medium.
Another experiment was then set up with a modified Reese's medium,
in which casamino acids was substituted for protepeptone. The experiment
is summarized in Table XX. The data show that the modified Reese's medium
gives equal or better protein yields when made up with tap water than
when made up with distilled water. Surprisingly, the best protein yields
were obtained from this modified medium from which the MgSO,-7 H_0, CaCl™
and trace elements^ had been omitted, showing that sufficient Mg, Ca, and
trace-element minerals were available from the tap water and the paper,
and in such proportions as to give even better results. Again it was shown
that the addition of urea gave a good improvement in protein yield, while
the addition of casamino acids gave a lesser improvements.
Effect of Type of Newspaper
Up to this time most of our work was done with ball-milled Wall
Street Journal. Since the Denver Post became available to us in shredded
form, we carried out an experiment to compare the yield on these two
ball-milled materials, using fungi P9, PlO and P26 and Medium G supplemented
with trace elements and urea. The data are given in Table XXI. The 2-day
vegetative seed media (Modified Reese's with casamino acids) gave higher
protein yields from the Wall Street Journal, but the 5-day harvest flasks
gave slightly higher yields from P9 and PlO on the Denver Post, but from
P26 on the Wall Street Journal.
Effect of Heating Newspaper
12
Since Ghose has found that heating cellulose to 200 C for 20
minutes gives a substrate providing improved cellulase synthesis by
Trichoderma viride, we tried the process on newspaper (Wall Street Journal).
After heating and ball-milling the paper was incorporated into medium G
supplemented with trace elements and urea, and fermented with fungus cultures
P9, PlO, and P26. As indicated in Table XXII, the three fungus cultures
all gave slightly higher protein yields on the heated paper.
The experimental work carried out thus far indicates that Reese's
medium, prepared either with protopeptone or casamino acids as a nutrient
supplement, is superior for growing the vegetative seed. In the case of
cultures P9 and PlO, protopeptone or casamino acids stimulates the production
of large numbers of conidiospores. An experiment was carried out substituting
disodium ethylenediamine tetraacetic acid for protopeptone in Reese's medium.
-10-
-------
The data, in Table XXIII show that it does stimulate both conidiospore
formation and protein synthesis, although not as well as protopeptone.
Thus, the effect is probably at least partly a chelation phenomenon rather
than a nutritional one.
A large experiment was then set up in order to establish the best
combination of ingredients for protein synthesis and cellulose utilization
from ball-milled newspaper in media based on Reese's medium and Medium G.
Table XXIV gives the data. In general, the best yields came from P9 and
P10 in Medium G-based media supplemented with urea and yeast autolysate
or casamino acids. Culture P26 produced little or no protein in the
Medium G-based media, but gave good yields in nearly all of the Reese's
medium variations. There were striking differences in terminal pH. Nearly
all of the Reese's medium variations gave low pH values. The range was
pH 2.9 to 7.0, with more than half the cultures between 2.9 and 3.6. The
Medium G variations ranged from pH 3.6 to 7.8, with more than half at
7.0 to 7.8. Those flasks with high urea concentration (0.06%) gave high
pH values. Because of a malfunction in a temperature regulator on one
of the shaker-incubators, 22 of the 140 flasks were incubated at temperatures
up to 37 C. The protein yields in these flasks were generally low. Protein
yields were also low in cultures with terminal pH values above 7.4.
Shaking Rate
An experiment was carried out to determine the influence of shaking
rate on protein synthesis. The three rates employed were 175, 264 and
350 RPM. The three fungus cultures P9, PlO and P26, were grown on Reese's
medium, supplemented with casamino acids instead of protopeptone, and
2% ball-milled Denver Post. The data, given in Table XXV indicate little
difference. The two lower rates of shaking give slightly higher protein
yields than the high rate for cultures P9 and P26, and the same yield for
PlO, within experimental error.
Concentration of Paper and of Nitrogen Source
An experiment was then carried out to evaluate the effect of paper
concentrations. (2, 4, 8 and 16 g per 100 ml of modified Reese's medium)
and nitrogen source (1.4 g/1 vs. 14 g/1 of ammonium sulfate). The data,
given in Table XXVI indicate that protein yield increases with increasing
paper concentration up to 8 g/100 ml at a nitrogen concentration of 1.4
g/1 of ammonium sulfate, and then drops off at 16 g/1. At 14 g/1 of
ammonium sulfate, protein yields are higher in most cases, particularly
in the flasks containing 16 g/100 ml of paper.
A similar experiment was set up using modified medium G. The results,
given in Table XXVII, were distinctly different, in that the protein yields
were larger in the low nitrogen concentration (10 g/1) than in the high
(50 g/1), in most cases. Yields generally increased with increasing paper
concentration up to 16 g/100 ml, but were less than proportional to paper
concentration, i.e. 16 g never gave 8 times the protein yield of 2 g.
-11-
-------
Effect of Tween-80
Since Reese and McGuire13 have reported that the presence of Tween-80
in the culture medium increases the rate of cellulase production by
Trichoderma viride. the effect of this surfactant (sorbitan monooleate)was
investigated by incorporating it into Reese's medium and medium G containing
2% newspaper at levels of 0.01, 0.1 and 0.5 g/1. The data, given in Table
XXVIII, indicate slight stimulation of 5-day protein yield at the two lower
levels in Reese's medium, but slight inhibition at the highest level. It
is interesting to note that Tween-80 had no stimulatory effect on protein
yield in medium G, but did exhibit a slight inhibitory effect at the highest
level as it did in Reese's medium.
Since heated and ball-milled newspaper was shown (Table XXII) to
give higher protein yields than unheated ball-milled newspaper, it was hoped
that heating alone might improve protein yields to the point where ball-
milling might be unnecessary. In order to evaluate this point, a comprehensive
experiment was set up using medium G as well as medium G supplemented with urea &
with urea and yeast autolysate, along with either coarsely shredded newspaper,
shredded and heated newspaper, or ball-milled newspaper. First a series was
harvested and analyzed after 5 days' incubation. The data, given in Table
XXIX, indicate much higher protein yields from ball-milled newspaper. In
view of the possibility that slower growth of the fungi on shredded paper
might be responsible for the results, the entire experiment was repeated,
with 3 times as many flasks; one series was analyzed after 5 days, a second
after 9 days and a third after 14 days. The data, given in the same Table,
do show considerably higher protein values after 14 days from the shredded
and shredded and heated paper, but again the yields from the ball-milled
paper are far superior, regardless of incubation time. The shredded-heated-
paper did not give significantly higher protein yields than unheated-shredded-
paper.
Scale-up Studies in Stirred-Jar Fermentors
It is well known in the fermentation industry that results obtained
in shake flasks are not necessarily duplicated in larger stirred-jar fermentors.
They do, however, serve as useful guides for such work, and many variables
such as pH, nitrogen sources, buffers and nutrients can be approximately
optimized in shake flask studies before proceeding to the more time-consuming
stirred-jar work.
Stirred-jar studies were carried out in a New Brunswick Scientific Co.
Model CMF 314 microferm floor fermentor consisting of three 14-liter
fermentors in a constant temperature bath. The fermentors were equipped
with automatic foam sensing and defoamant addition controls and automatic
constant - pH titration equipment. Figure 1 illustrates the equipment in
use. The entire fermentor, with culture media and all accessory devices and
tubing included, was autoclaved for 30 minutes at 20 psig for sterilization.
The volume of medium in each jar was 6.57 to 10 liters, and the temperature
was controlled at 30 C. Air was sterilized by filtration through a 15 cm
-12-
-------
by 2.0 cm column of packed sterile absorbent cotton. Samples were collected
every working day for analytical and pH determinations. Table XXX summarizes
the variables investigated in a total of 12 runs, and the final protein
yields and cellulose consumption. The first two runs were not included as
difficulties with temperature control and air filtration in these preliminary
runs gave erratic results. Results are also presented graphically in
Figures 2-19. Run 10 was not graphed because of solids- build-up problems.
A study of the Table and the figures leads to the following conclusions:
1. Culture P9, Myrothecium verrucaria. grows well on ball-milled
newspaper, actively consumes cellulose and synthesizes cell material
containing protein.
2. The optimum stirring rate for protein synthesis is 300 to 400 RPM,
higher stirring rates could not be employed because of excessive splashing
and foaming, leading to excessive build-up of solids on the walls of the
fertnentor above the normal liquid level.
3. The optimum aeration rate depends partially upon stirring rate,
as oxygen transfer increases with increased stirring rate. From 3 to 6 1/min
appears to be optimal at 300 to 400 RPM. At 100 RPM, 6 1/min is superior
to lower air rates. Again, very high air rates must be avoided because of
foaming and solids-build-up problems.
4. The pH optimum was broad, from 3.9 to 6.5.
5. The incorporation of 0.03% urea into the medium G prevented the
pH from dropping below pH 4.8, and maintained it within the optimum range.
Urea also increased the protein yield.
6. The incorporation of yeast autolysate in addition to urea greatly
stimulated both growth rate and protein yield.
7. The maximum rate of protein synthesis was obtained in run 11,
jar 1. A protein yield of 1.2 mg/ml was obtained in 4 days, at which time
54% of the cellulose originally present had been consumed. From these data,
the calculated rates of cellulose utilization and protein biosynthesis are:
Cellulose consumed, g/l/day 5.4
Protein produced g/l/day 0.3
Protein, 7<, yield as
% of cellulose consumed 5.6
8. The protein yield increases with increasing paper concentration up
to 8 g per 100 ml of medium, the highest level tested.
The highest final protein yields, 1.42 and 1.36 mg/1 (which are
probably the same, within experimental error) were obtained in stirred-jar
number 1 of run 12 and 3 of run 10 respectively, both in medium G3. Jar 3
of run 10 was at the highest air rate employed, 6.0 1/min, and the highest
stirring rate, 400 RPM at the start, reduced to 300 RPM after 48 hours.
Thus, oxygen transfer rate, which is increased by both air rate and stirring
rate, is limiting growth. Higher air rates or stirring rates could not be
used because of problems encountered with foaming and solids build-up on the
walls of the fermentor above the normal liquid level. The solids build-up
problem in run 10 was so severe that a large proportion of the fermentor
solids were clinging to the walls of the fermentor; consequently it was
impossible to obtain satisfactory protein analyses on samples collected during
-13-
-------
tlu: run, and all of the protein analyses from day 3 through day 10 were
very low - in the range of 0.2 to 0.5 mg/ml. At the termination of the
experiment, the solids were scraped back into the fermentor, and thoroughly
mixed, so that the final analyses would be accurate.
Run 12 clearly indicated the beneficial effect of the yeast autolysate
in the medium G-3 on protein synthesis. In general high protein yield,
and high rate of protein synthesis correlated well with cellulose utilization.
It is interesting to speculate whether better results could be
obtained at higher substrate concentrations. Runs 8, 9 and 10 clearly
indicate that the oxygen transfer rate is limiting protein synthesis at
4 g of ball-milled newspaper per 100 ml of medium. Oxygen demand increases
with increasing substrate concentration, as does the viscosity of the medium.
The viscosity increase will decrease the oxygen transfer rate. Thus, it is
possible that further increases in the substrate concentration might not give
higher protein yields or higher rates of protein synthesis. Jar 3 of run 7,
where 8 g of substrate per 100 ml of medium was employed, is a case in point.
Even after 24 days' incubation the protein yield was only 0.63 mg/ml,
probably as a result of insufficient oxygen transfer rate.
Composition of Final Product
In order to evaluate the final product from run 10, the fermentor
contents of Jar 3 were evaporated to dryness by boiling, with stirring, on
an electric hot plate in a 5 1 stainless steel beaker. When the total
volume was reduced to 1-2.1, the material was placed in a pyrex glass dish,
and evaporated to dryness in an oven at 90-100 C. The dried cake was then
ball-milled into a fine powder for analysis.
Moisture content was determined by the A.O.A.C. method, p.327
after equilibration of the ball-milled material with the atmosphere at
ambient temperature and humidity for several hours. Ash content was determined
gravimetrically by ignition in a Bunsen burner flame.
A suspension of this material, 1 g per 100 ml, was prepared and an
aliquot analyzed for total protein by the "revised" Biuret method. Following
centrifugation of this suspension, aliquots of supernatant were analyzed
for soluble protein in a similar manner.
Ammonia nitrogen and organic nitrogen were estimated by the A.P.H.A.
method, pages 240-242 and A.O.A.C., page 1617 respectively. Total Kjeldahl
nitrogen was calculated as the sum of the ammonia and organic nitrogen values.
Nitrate nitrogen was determined colorimetrically by a brucine reaction .
Nitrite nitrogen was determined colorimetrically by a diazotization method^.
Total carbohydrates were determined on a sample of the material
dissolved in 67% H^SO, by the anthrone reaction^. Soluble carbohydrates
-14-
-------
were measured similarly on a centrifuged sample of the material. Cellulose
content was estimated according to the method established in this laboratory^.
Total lipids were estimated by the A.O.A.C. method, pages 197-19817. The
data are summarized in TableXXXI, along with similar data on corn and wheat20,
both of which are excellent feed materials for cattle, although slightly
low in protein to make a complete food for human consumption. It can be
seen that our material is higher in ash and cellulose, and somewhat lower
in protein. It is likely that these factors would not limit its use as
a cattle feed, however.
Discussion
The screening phase of this prp^&et reexamined the occurence of
rapidly-growing cellulolytic microorganisms in nature. The methods used
deliberately selected only those organisms which were capable of a high
rate of protein synthesis in submerged culture. As might have been expected,
the organisms isolated were from genera long known to be actively cellulolytic,
Myrothecium verrucaria, Trichoderma viride, Aspergillus fumigatus and
Cellulomonas. As pointed out by Gascoigne and Gascoigne''-, the 19fhcentury
biochemist, Hoppe-Seyler, had studied cellulase production by fungi, and
the 19th century mycologist, DeBary described several cellulolytic fungi.
In our experiments , Myrothecium verrucaria produced the highest
yields of protein, and hence our optimization work was carried out with _,
this organism. This is in agreement with many reports in the literature
emphasizing the very active cellulolytic properties of this fungus.
By comparison with'cellulose, lignin is very slowly attacked by
microorganisms^, and it is probable that little or no lignin was consumed
in any of our experiments, although this cannot be stated with certainty,
since direct analyses for lignin were not carried out.
Our data are not sufficient to make a material balance, but some
hypothetical calculations of this type may be given for jar 1 run 12, which
gave the highest protein yield.
Paper originally present, g/1 40
Protein, g/1 (from biuret analysis) 1.42
Protein, g/1 (from organic N x 6.25) 3.3
Cellulose original present, g/1 20.4
Cellulose consumed, g/1 12.7
Assume that 50% of the 12.7 g of cellulose substrate consumed was
oxidized for energy, and 50% was assimilated and converted to cell material
which contains 30% protein. (These values were taken from Gray11 as being
fairly typical for fungi imperfecti grown on glucose.) Then the amount
of cellulose converted to cell material would be 6.35 g/1 and the protein
would be 1.91 g/1. Gray determined protein as organic N x 6.25. Thus,
it can be seen that our corresponding protein value, 3.3 g/1, is consider-
ably higher than the example given by Gray. The true protein value of our
product (as well as Gray's) is probably closer to the biuret value of
1.42 g/1, since NX 6.25 includes in addition to true protein all other
nitrogen compounds found in the cells such as amino acids, phospholipids,
purines and pyrimidines DNA and RNA. In any event, returning to our
-15-
-------
product calculations beginning with 40 g/1 of newspaper, it should be
possible to produce 33.7 g/1 of final product containing 3.3 g/1 of
"protein" calculated as N x 6.25.
The work accomplished was nearly sufficient to permit the design
and construction of a pilot plant, although a few more stirred-jar
runs using higher concentrations of ball-milled paper and perhaps higher
concentrations of ammonium phosphate and urea may be desirable. Although
Ciegler and Lillhoj^S dp not list tjtyrotfrecium verrucaria among the
mycotoxin-producing fungi, some preliminary feeding studies with mice
and rats on our final products would be highly desirable before proceeding
with pilot plant construction in order to obtain some evaluation to
toxicity and dietary value.
-16-
-------
Table I
The Utilization of Cellulose by Enrichment Cultures
Serial
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Culture Medium
No. a«Newspaper
b-Purif led
cellulose
c-Hew^print
(lg/100 ml)
353x114-30 Pi a
353x114-30 PI c
353x114-30 SM a
353x114-30 PI a
353x99-B SM a
353x99-B SM b
353x99-8 SM c
353x113-8 SM b
353x113-10 SM a
353x113-10 SM c
353x113-11 SM a
353x113-11 SM b
353x113-11 SM c
353x113-15 SM a
353x113-15 SM b
353x113-15 SM c
353x113-19 SM b
353x119-19 SM c
353x113-24 SM a
353x113-24 SM b
353x113-24 SM c
353x113-25 SM a
353x113-25 SM b
353x113-25 SM c
353x113-8 Reese a
353x113-8 Reese b
353x113-8 Reese c
353x113-10 Reese a
353x113-10 Reese b
Medium Cellulase
Enrichment Assay
M=Mineral
salts
E=0rganic
nutrients
HC=Hydrocarbon
enrichment
M
M
E
M
E +
E +
E +
E
E +
E •+
E +
E
E
E
E
E
E
E
E
E +
E +
E +
E +
E
E
E
E +
E +
E +
Final Cell
Count per ml
8.9 x 107
4.3 x 107
3.2 x 109
7.3 x 107
3 x 106
3 x 106
3 x 106
2.2 x 109
1.1 x 108
4 x 106
1.0 x 109
5.5 x 109
7.7 x 109
2.4 x 109
4.6 x 109
5.7 x 109
1.9 x 109
1.2 x 109
1.6 x 109
4.1 x 109
5.9 x 109
4.5 x 109
8.1 x 109
6.8 x 109
5.4 x 109
1.9 x 109
1.3 x 109
1.7 x 108
9 x 106
Protein
mg/ml
(Biuret)
1.38
0.29
0.35
0.62
0.53
0.75
0.58
0.25
0.53
0.66
0.51
0.55
0.44
0.20
0.28
0.34
0.22
0.31
0.39
0.37
0.36
0.56
0.36
0.55
0.60
6.28
0.28
0.75
0.85
% of
Cellulose
Utilized*
31
4
4
X
54
59
56
3
52
59
45
27
19
6
5
25
9
4
34
5
17
24
14
28
X
36
23
X
81
-17-
-------
Table I (cont.)
Serial Culture * Medium
No. No. a-Newspaper
b=Furified
cellulose
c=Newsprint
30
31
32
33
34
35
36
37
38
39
AO
41
353x113-10
353x113-11
353x113-11
' 353x113-15
353x113-18
353x113-24
353x113-24
353x113-25
353x113-25
353x113-30
353x113-30.
353x113-30
Reese c
Reese a
Reese b
BH a
BH c
BH a
BH b
BH a
BH b
SM a
SM b
SM c
Medium
Enrichment
M=Mineral
snlts
E=0rgnnic
nutrients,
E
E
E
M
M
M
M
M
M
E
E
E
Cellulose Final Cell
Assay Count per ml
+ 4 x 106
4.6 x 109
TOTC
1.4 x 109
2.7 x 108
l.lxlO10
8.1 x 109
3.2 x 109
1.0 x 1010
+ 1.1 x 109
1.1 x 109
1.6 x 109
Protein
mg/ml
(Biuret)
0.59
0.38
0.98
0.23
0.36
0.44
0.39
0.40
0.48
0.47
0.23
0.21
7. of
Cellulate
Utilized
65
X
14
X
29
20
20
34
15
20
3
7
X = Analysis not done or not included because of known error.
* As % of original cellulose in sterile control from 1 g of ccllulosic substrate in
100 ml of culture medium.
SM o Srinivasan's medium.
BH = Bushnell-Haas medium.
-18-
-------
Table II
i
i-»
vo
The Utilization of Cellulose by Pure Cultures
Serial
No.
i
2
3
4
5
6
7
8
9
10
Culture
No.
P 9
P 9
P 10
353x85-1
353x85-1
353x85-1
353x99-B
353x99-8
« 353x99-8
C 264
Med ium
a=Newsprint
b= Purified
cellulose
c=NewsDaDP.r
(lg/100 ml)
SM a
SM c
SM c
SM a
SM b
SM c
Reese .1
Reese b
Reese c
SM a
Medium
Enrichment
M-Mineral salts
E^-Organic
nutrients
,
E
E
E
E
E
E
E
E
E
E
Celluiase Final
Assay Cr-11
Count
per ml
+ TNTC
+ 8.1 x 107
+ 7.6 x 107
+ 4.0 x 106
+ 6.0 x 106
+ 2.0 x 106
-1- 9.0 x 106
+ 3.0 x 106
+ 1.4 x 106
X Heavy
fungus
gnrowth
Protein
mg/ml
(Biuret)
0.52
0.26
0.28
0.22
0.49
0.40
0.81*
0.84r
0.32
0.44
7. .of
Cellulose
Utilized
47
30
8
X
X
X
54
78
34
X
* Biuret value not corrected for blank
-i- Biuret value too high because of interfering turbidity
X Analysis not done or not included because of known error
-------
•Table III.
Characteristics of
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
Culture No.
353 x 113-30
*
353 x 99-B
*
353 x 113-8
353 x 113-10
353 x 113-11
353 x 113-15
353 x 113-18
353 x 113-19
353 x 113-24
353 x 113-25
P 9
P 10
353 x 85-1
353 x 99-B*
4.
C 264*
Ccllulolvtic Cultures from Tables I and II
Pure (P) or Serial No.
Enrichment (E)
E
E
E
E
E
E
E
E
E
E
P
P
P
P
P
1,2,3,4,39,40,41
5,6,7
8,25,26,2,7
9,10,28,29,30
11,12,13,31,32
14,15,16,33
34
17,18
19,20,21,35,36
22,23,24,37,38
1,2
3
4,5,6
7,8,9
10
Source
Soil SI
Aerobic sewage
sludge
Soil 55
Soil 58
Aerobic sewage
sludge
Soil 49
Soil 52
Soil 53
Aerobic sewage
sludge
Anaerobic
sewage
s ludge
Soil
No. 53
Soil
No. 53
Compost soil
No. 59
Aerobic
sewage
sludge
HC ox.+
Description
Mixed bacteria
Fungus
Mixed bacteria
n n
n n
ii n
"
n n
n ii
Fungus, identified
as Myrothecium
verrucaria
Same
Fungus, identified
as Aspergillus
fumigatus
Fungus, identified
as Trichoderma
Fungus, identified
as Aspergillus
+ Isolated on the hydrocarbon, n-hcxadecane
* After one series of fermentations, this culture was established to be pure.
fumigatus
-20-
-------
Table IV
The Optimization of Conditions for Growth
Protein Synthesis and Cellulose Utilization by
Serial Culture Med.+
No.
1
2
3
4
5
6
7
TV
TV
TV
SM
SM
SM
TV
8 P-9 TV
9
10
11
12
13
14
15
16
17
TV
SM
SM
SM
TV
TV
TV
SM
SM
19 V SM
19
20
21
22
23
24
25 P
26
27
28
29
TV
TV
TV
SM
SM
SM
10 TV
TV
TV
SM
SM
30 V SM
Pure Cultures
Temp.
30
37
45
30
37
45
30
37
45
30
37
45
' 30
37
45
30
37
45
30
37
45
30
37
45
30
37
45
30
37
45
of Fungi
on 1% Newsprint
Time Micro-
of Inc. , scopi£
Days
2
2
2
2
2
2
5
5
5
5
5
5
9
9
9
9
9
9
2
2
2
2
2
2
5
5
5
5
5
5
Exam.
a3+
a3+
No growth
a3+
a3+
No growth
a3+
a3+
No growth
a 3+
a2+
No growth
a3+
a3+
No growth
a 3+
a3+
No growth
a3+
a3+
No growth
a 3+
a3+
No growth
a 3+
a3+
No growth
a3+
a3+
No growth
pH
4.8
4.8
5.3
3.6
4.7
5.7
4.4
5.5
5.9
3.0
3.6
5.7
3.7
5.4
5.3
3.0
3.9
5.7
5.3
5.3
5,6
3.3
4.1
5.7
4.2
5.3
5.7
3.1
3.7
5.8
Protein I Cellulose
nig /ml Decomposed
0.41
0.31
0.04
0.22
0.15
0.03
0.48
0.37
0.06
0.45
0.34
0.08
0.41
0.31
0.12
0.48
0.28
0.08
0.39
0.27
0.03
0.33
0.35
0.05
0.49
0.38
0.10
0.44
0.34
0.07
59
70
-
45
38
-
75
75
-
60
65
-
77
84
-
54
49
-
74
69
-
44
50
-
75
81
-
48
55
-
-21-
-------
Serial Culture Hed.*
Ho.
31 P-10 TV
32
33
34
35
36
TV
TV
SH
SM
, SM
37 353x85-1 TV
38
39
40
41
42
43
44
45
46
47
48
49
SO
51
52
53
54
TV
TV
SM
SM
SM
TV
TV
TV
SM
SM
SM
TV
TV
TV
SM
SM
SM
S3 353x998 TV
5i
57
58
59
60
61
62
63
64
TV
TV
SM
SM
SM
TV
TV
TV
SM
65 v at
Table IV (cont.)
Temp .
30
37
45
30
37
45
30
37
45
30
37
45
30
37
45
30
37
45
30
37
45
30
37
45
30
37
45
30
37
45
30
37
45
3U
37
Time
of Inc.
Days
9
9
9
9
9
9
2
2
2
2
2
2
5
5
5
5
5
5
9
9
9
9
9
9
2
2
2
2
2
2
5
5
5
5
5
Micro*
, scop if
Exam.
a3+
a 3+
b2-t-
a3+
3+
No growth
b3+
b3+
b3+
b3+
b3+
b3+
b3+
b3+
b3+
b3+
b3+
b2+
b3+
b2+
b3+
b2+
b2+
b2+
b3+
No growth
No growth
b3+
b2+
No growth
b3+
b2+
b2+
b3+
b3+
PH
4.0
5.3
5.6
3.2
4.2
5.7
6.3
6.5
5.4
5.8
5.1
4.9
5.2
5.5
5.0
4.8
5.1
4.6
5.4
5.4
6.2
4.9
5.2
5.8
3.9
6.1
5.7
2.*
6.0
5.6
5.5
6.1
5.3
3.1
4.5
Protein
ag/mi
0.35
0.29
0.19
0.43
0.30
0.08
0,04
0.11
0.27
0.06
0.25
0.25
0.26
0.40
0.15
0.19
0.34
0.29
0.36
0.31
0.13
0.40
0.36
0.28
0.31
0.01
0.03
0.48
0.04
0.05
0.35
0.12
0.07
0.45
0.19
X Cellulose
Decomposed
76
85
61
54
61
-
-
-
52
-
16
49
59
70
-
-
66
72
78
80
81
77
80
81
62
-
-
58
-
-
75
.
.
71
.
-22-
-------
Table IV (cont.l
Serial Culture Med.
No.
M 353x998 SM
•7
68
69
70
71
«
73 C-2
74
75
76
77
78
19
80
Bl
82
83
84
65
66
67
S«
89
$0
TV
TV
TV
SM
SM
SM
!64 TV
TV
TV
SM
SM
SM
TV
TV
! TV
SM
SM
SM
TV
TV
TV
SM
SM
SM
*i Sterile Control TV
92
»3
tt
>5
Hi
•7
$8
f§
too
JL01 '
TV
TV
SM
SM
SM
TV
TV
TV
SM
SM
Temp.
45
30
37
45
30
37
45
30
37
45
30
37
45
30
37
. 45
30
37
45
30
37
45
30
37
45
30
37
45
30
37
45
30
37
45
30
37
Time
of Inc.
Days
5
9
9
9
9
9
9
2
2
2
2
2
2
5
5
5
5
5
5
9
9
9
9
9
9
2
2
2
2
2
2
5
5
5
5
5
Micro -
, scoplj
Exaa.
b24
b3+
b3+
No growth
b2+
b3+
b2+
b3+
b3+
b3+
b3+
b3+
b3+
b3+
b3+
b3+
b3+
b3+
b2+
b3+
b3+
b+
bf
b2-f
b+
Mo growth
PH
5.5
5.6
5.9
5.6
3.2
5.5
5.1
5.3
5.3
5.8
4.0
4.6
5.1
5.3
5.4
6.2
4.4
4.4
5.8
5.4
6.0
6.5
5.0
5.5
5.9
5.5
5.4
5.6
5.5
5.6
5.6
5.6
5.7
5.7
5.5
5.5
Protein
m&/Ml
0.05
0.35
0.05
0.07
0.45
0.16
0.29
0.23
0.38
0.32
0.27
0.23
0.21
0.28
0.31
0.20
0.28
0.31
0.26
0.26
0.38
0.13
0.31
0.37
0.23
-
-
- •
-
-
-
-
-
-
~
% Cellulose
Decomposed
.
83
.
68
.
76
38
63
65
49
39
61
73
77
-
64
61
72
84
82
-
77
79
82
-
-
-
-
-
.1
•
-
-
••
-23-
-------
Table jy (cent.)
Serial Culture Med.
Mo.
102 Sterile Control SM
103
104
105
106
107
108
TV
TV
TV
SM
SM
SM
Temp.
45
30
37
45
30
37
45
Tine Micro- pH Protein
of Inc. , scopic mg/Jnl
Days Exam.*
X Cellulose
Decomposed
5 No growth 5.6 -
9
9
9
9
9
9
5.4
5,4
5.4
4.3
4.9
5.3
-
-
-
-
-
-
* Microscopic examination:
a ». mostly oval yeast-like cells
b » Filamentous mycelium
+ Media;
TV = Reese's medium
SM = Srinivasan's medium (See Appendix)
Each flask was inoculated with 1 ml of a 48-hour shake flask culture, grown as follows:
P-9 and P-10 SM medium + 17. newsprint, 30 C
353x85-1 TV medium + 17, newsprint, 45 C
353x99-B TV medium + 17. newsprint, 30 C
C-264 SM medium + 1% newsprint, 37 C
-24-
-------
Table V
The Influence of Paper Concentration on
the Growth of Fungi
Serial
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Culture
P-9
P-9
P-9
P-10
P-10
P-10
353x998
353x998
353x998
Controls
Controls
Controls
P9
P-10
353x998
P-9
P-10
353x998
Control
Control
Med,+
A
8
C
A
B
C
A
8
C
•A
B
C
D
D
D
E
E
E
D
E
Microscopic
2 days
« *
Examination
5 d.iys
No growth Fair growth
Good growth
No growth
No growth
Good growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
Good growth
Good growth
No growth
No growth
No growth
No growth
No growth
No growth
Good growth
Good growth
Fair growth
Good growth
Fair growth
Fair growth
Fair growth
No growth
No growth
No growth
No growth
Good growth
Good growth
No growth
Fair growth
Good growth
Good growth
No growth
No growth
pH
2.6
2.6
2.6
2.7
2.5
2.6
3.4
2.3
3.7
3.9
4.7
3.7
6.5
6.3
6.3
5.9
5.6
6.6
4.9
4.6
* All cultures were examined at 2 days' and 5 days' incubation.
Incubated at 30 C
Inoculum: Growth from a nutrient agar slant.
+ Media: A 30 g Newspaper, ball-milled (17% by weight paper)
All media B 30 g Purified cellulose (17% by weight cellulose)
were made C 30 g Newsprint, ball-milled (17% by weight paper)
from 150ml D 1.5 g Newsprint, blended (0.997. by weight paper)
of Reese's E 2.0 g Newsprint, blended (1.37. by weight paper)
medium,with
the following
additions
-25-
-------
Table VI
Simplification
of Culture Media for
Paper Digestion and Protein Synthesis
Serial
Mo. Culture Med.+
1 P-9 F
2
3
4
5
6
7
81
9
10 P
11
12
13
14
15
16
17
'
19 353
20
21
22
23
24
25
26
27
28 Con
29
30
31
32
33
G
SM
F
G
SM
F
G
-10 F
G
SM
F
G
SH
F
G
SBT
(99B F
G
SH
F
G
SM:
F
G
SMf
trol F
G
SMI
F
G
SM
Inc.
Time,
days
2
2
2
5
5
5
9
9
9
2
2
2
5
5
5
9
9
9
2
2
2
5
5
5
9
9
9
2
2
2
5
5
5
Microscopic
Exam.
Fair growth
Good "
Good "
Fair "
Fair "
Good "
Good "
Good "
Good "
Fair "
Fair "
Pair "
Fair "
Good "
Good "
Fair "
Good "
Good "
Good "
Good "
Good "
Good "
Fair "
Good "
Good "
Good "
Good "
None
PH
3.1
4.9
2,8
2.8
3.3
2.5
2.9
3.9
2.5
3.1
5.4
3.5
2.8
3.2
2.4
2.7
3.4
2.6
2.5
2.8
2.2
2.5
2.9
2.6
2.7
3.3
2.5
4.1
5.3
5.7
4.2
5.4
5.7
Protein
mg/ml
V*^^B^>HA"^^X*^BIIBB'BBH
0.06
0.13
0.37
0.52
0.75
0.55
0.44
0.83
0.49
0.10
0.10
0.18
0.37
0.69
0.57
0.52
0.90
0.42
0.39
0.68
0.65
0.35
0.60
0.28
0.41
1.08
0.52
-26-
-------
Table VI (cont)
Serial
No. Culture
34 Control
35
36
Med.H-
F
G
SM
Inc.
Time,
days
9
9
9
Microscopic
Exam.
None
1
pH
4.3
5.3
5.6
Protein
mg/ml
-
-
_
+ Media: F 100 ml tap water + 1 g (NH,),SO, -f 2 g of balled-milled newspaper
(Wall St. Journal) <* * <*
G 100 ml tap water + 1 g (NH4)2HP04 + 2 g of ball-milled newspaper (WSJ)
SM 100 ml of Srinivaaan medium + 2 g of ball-milled newspaper (WSJ)
Inoculum: 5 day old shake flask cultures from Table V. (Reese's medium plus
17% newsprint)
Flask 3 - Inoculum for cultures 1-9
Flask 6 - Inoculum for cultures 10-18
Flask 7 - Inoculum for cultures 19-27
-27-
-------
Table VII
Growth and
Protein Synthesis by
Cellulolytic Fungi Growing on Medium H
Flask No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Inoculum:
Inoculum Temp.°C Time, Days Terminal pH
P9 30
„
M ii
P10
it ii
ii M
P26 "
II II
II II
C264 37
II II
11 M
353x85-1 ' 45
M ||
II M
Control 30
11 37
45
30
37
45
30
37
45
1 ml of a 9 day shake
2
5
9
2
5
9
2
5
9
2
5
9
2
5
9
2
2
2
5
5
5
9
9
9
flask culture from the
3.5
3.4
3.6
3.5
3.3
4.1
3.4
3.8
3.7
2.9
3.1
3.7
3.4
3.1
3.1
4.8
4.8
4.8
4.9
4.8
4.8
5.2
5.2
5.2
exoerimen
Protein mg/ml
0.11
0.15
0.30
0.13
0.20
0.33
0.15
0.11
0.07
0.43
0.28
0.31
0.18
0.19
0.30
-
-
-
-
-
.
_
_
-
t of
Table VI, growing in Medium G with 27, ball-milled
Wall Street Journal.
-28-
-------
Table VIII
NO
VO
Characteristics of Cellulolytic Bacteria
Tube
I
2
3
4
5
6
No. Inoculum
Strip
X125-1
X125-2
X125-3
X125-4
Cellulomas sp.
Control
Filter Paper
Test
5 das 14 das
+ 3+
+ 3+
+ 3+
+ 3+
+ 3+
0 0
CMC ^ Agar Stroke
hydrolysis
5 das
3+ Filiform
3+ "
3+
3+ "
3+ "
0 "
Nut. broth Methyl Vogues Indole
Red Pros sauer
Mod.turb. Neg. Neg. Neg.
n n n n
n ti n n
n n n n
n n n n
n n M n
CMC medium was prepared by adding 4.9% of sodium carboxymethyl cellulose (Hercules type 7MF).
A 3+ reaction indicates complete liquefaction of the entire tube of viscous medium. 3+ on
the filter-paper strip test means dissolution of the paper into a small amount of fibrous material.
All were small Gram variable, non-motile rods which did not ferment glucose.
-------
Table IX
Growth and Protein Synthesis by Cellulomonas and Related
Bacterial Cultures Growing on Ball-Milled Paper or
Purified Cellulose in Srinivasan's Medium Incubated at 37 C.
Flask No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Inoculum Inc. time Substrate +
days
353x125-1 5 A
353x125-2
353x125-3
353x125-4
Cellulomonas
353x,25-l
353x125-2
353x125-3
353x125-4
Cellulomonas
353x125-1
353x125-2
353x125-3
353x125-4
Cellulomonas
Control
ii
ii
A
A
A
A
B
B
B
B
B
C
C
C
C
C
A
B
C
/
Term
37 C
4.7
4.9
4.9
4.9
5.0
4.7
4.8
4.8
4.8
5.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
pH Protein Microscopic
nig /ml Exam.
30 C 37 C
.03
.03
.05
.03
.03
.07
.07
.07
.07
.07
1
. 02 Nothing
-.01
-.01
-.01
.01
-
-
+ Substrate:
A - Srinivasan's med. +2% ball-milled newspaper (Wall St. Journal)
B - Srinivasan's med. + TL purified cellulose
C - Srinivasan's medium + 2% newsprint
V
-30-
-------
Table X
Growth and Protein Synthesis by Trichoderma Vjride Grown
on Ball-Milled Newsprint in Reese's and
Srinivasan's Media at 30 and 37 C
*
Medium Inc. Term pH
Time, Days 30 C 37 C
-I
-2
-3
-4
-5
-6
-7
-3
-9
-10
-11
-12
b
a
b
a
b
a
b
a
b
a
b
a
2
2
5
5
11
9
2
2
5
5
11
9
6.5
6.5
6.0
2.7
• 4.9
2.8
5.0
5.9
4.9
5.9
6.8
5.9
4.6
6.7
6.2
6.7
6.5
6.6
5.2
5.7
5.2
5.7
5.1
5.6
Protein rag/ml Cellulose Utilized %
30 C 37 C 30 C 37 C
-0.03 -0.04
0.05 0
0.43 0.17 87%
0.54 0.03
0.46 0.29 90%
0.51 0
-
-
-
-
-
-
Media a Srinivasan's medium + 17. newsprint
b Reese's medium + 1% newsprint
-31-
-------
Table XI
Chemical-
H20
H20
NaOH
NaOH
H3P04
H3P04
H2S04
H2S04
NaHS03
NaHSO,
Chemical Treatment of Pulped
(Wall Street Journal)
Treatment Wt. of paper before
treatment, g
Hot
Cold
Hot
Cold
Hot
Cold
Hot
Cold
Hot
Cold
9.83
9.71
9.66
9.78
9.86
9.73
9.92
9.85
9.73
9.67
Newspaper
Wt. of paper recovered
after treatment , g
9.60
9.56
7.53
9.21
8.38
9.46
7.61
9.57
10.06
10.00
-32-
-------
Table XII
Growth and Protein Synthesis on Chemically Treated
Papers in Medium H in Five Days
Flask No. Culture No. Paper Treatment Temp.°C Term.pH Protein mg/ml
1 P9
2 ' n
3 "
4 "
5 "
6 "
7 "
8 "
9 "
10 "
11 P10
12 "
13 "
14 "
15
16 "
17 "
18 "
19 "
20 "
21 353x85-1
22 "
23 "
24 "
25
26
27
28
29
30 "
31 P26
32
33
H20 Hot
H20 Cold
NaOH Hot
NaOH Cold
H3P04 Hot
H3P04 Cold
H2S04 Hot
H2S04 Cold
NaHS03 Hot
NaHS03 Cold
H20 Hot
H20 Cold
NaOH Hot
NaOH Cold
H3P04 Hot
H3P04 Cold
H2S04 Hot
H2S04 Cold
NaHS03 Hot
NaHS03 Cold
H20 Hot
H20 Cold
NaOH Hot
NaOH Cold
H3P04 Hot
HV°4 Cold
H2S04 Hot
H2S04 Cold
NaHS03 Hot
NaHS03 Cold
H20 Hot
H20 Cold
NaOH Hot
30
n
n
M
ii
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
45
n
45
1!
II
II
II
II
II
II
30
ii
ii
3.9
4.1
4.2
4.1
4.1
3.8
4.5
3.9
3.8
3.8
3.9
4.0
4.6
4.3
4.0
4.0
4.5
4.0
3.9
3.8
3.8
4.0
4.6
4.2
4.0
3.9
4.6
4.0
3.9
3.9
4.5
5.0
5.1
0.09
0.03
0.05
0.06
0.09
0.08
0.03
0.11
0.08
0.08
0.11
0.05
0.03
0.07
0.09
0.08
0.06
0.11
0.07
0.09
0.13
0.07
0.08
0.08
0.13
0.09
0.02
0.10
0.07
0.09
0.03
0
0.02
-33-
-------
Table XII (cont.)
Growth and Protein Synthesis on Chemically Treated
Papers in Medium H in Five Days
Flask No. Culture No. Paper Treatment Temp. C Terjn.pH
34 P26
35
36 "
37 "
38 "
39 "
40 "
41 C-264
42 •'
43 "
44
45 "
46 "
47 "
48
49 "
50 "
51 Control
52 "
53 "
54 "
55 "
56
57 "
58 "
59 "
60 »
61 Trichoderma viride
62 "
63
64 »
65 "
66 "
NaOH Cold
H3P04 Hot
H3P04 Cold
H2S04 Hot
H2S04 Cold
NaHS03 Hot
NaHS03 Cold
H20 Hot
H20 Cold
NaOH Hot
NaOH Cold
H3P04 Hot
H3P04 Cold
H2S04 Hot
H2S04 Cold
NaHS03 Hot
NaHS03 Cold
H20 Hot
H20 Cold
NaOH Hot
NaOH Cold
H3P04 Hot
H3P04 Cold
H2S04 Hot
H2S04 Cold
NaHS03 Hot
NaHS03 Cold
H20 Hot
H20 Cold
NaOH Hot
NaOH Cold
H3P04 Hot
H3P04 Cold
30
n
n
ti
n
n
n
37
n
n
n
n
M
n
n
n
n
30
"
n
n
n
n
n
ii
n
"
n
"
n
n
M
n
4.9
4.4
4.5
5.2
4.7
4.2
4.4
4.2
4.0
4.2
4.9
3.9
3.8
4.4
4.0
3.9
3.8
5.2
5.3
5.4
5.2
5.1
5.2
5.2
5.2
5.1
5.1
5.2
5.1
5.1
5.2
4.8
5.1
Protein mg/ml
0.03
0.04
0.03
0.02
0.06
lost
0.02
0
0
0
0
0
0
0
0
0
0
-
-
-
-
-
-
-
-
-
-
-0.02
-0.06
0.02
0.02
0.05
0.01
-34-
-------
Table XII (cont.)
Growth and Protein Synthesis on Chemically Treated
Papers in Medium H in Five Days
Paper Treatment
Flask
67
68
69
70
71
72
73
74
75
76
77
No. Culture No.
Trichoderma viride
it
ii
ii
P9
P10
353x85-1
P26
C-264
Trichoderma viride
Control
Inoculum: 5 -day culture
Temp.°C
H2S04 Hot
H2S04 Cold
NaHS03 Hot
NaHS03 Cold
Ball-milled
ti
30
Term. pH
5.0
5.1
4.9
4.2
3.7
3.7
4.2
5.1
4.2
3.6
5.2
newspaper
Trichoderma
Protein mg/ml
-0.03
-0.01
-0.02
-0.01
0.14
0.15
0.10
-0.01
0.12
C.ll
-
from
viride
which was a 5-day culture in Srinivasan's medium with 17»
ball-milled newsprint.
Harvest: 5-day cultures in Medium H plus 1% ball-milled
Wall Street Journal.
-35-
-------
Table XIII
The Influence of the Growth Medium for the Inoculum
(Reese's Medium or Medium G) on Protein Yields
from Cultures Grown on Media G and H
Flask No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Inoculum Med.
G
G
R
R
G
G
R
R
G
G
R
R
Control
Control
Culture
P9
P9
P9
P9
P10
P10
P10
P10
P26
P26
P26
P26
None
None
Harvest
Flask Med.
G
H
G
H
G
H
G
H
G
H
G
H
G
H
Term.
PH
5.4
3.4
3.2
3.4
5.4
3.8
3.3
3.4
3.4
3.4
3.2
3.6
5.5
5.1
Protein, mg/ml
0.13
0.57
0.59
0.59
0.08
0.16
0.67
0.63
0.23
0.29
0.35
0.17
-
_
Inoculum; 2-day cultures in medium G or Reese's medium with 2%
ball-milled Wall Street Journal.
The inoculum cultures in Reese's medium showed much heavier
growth under the microscope than those in Medium G.
Harvest Flasks; 5-day cultures in medium G or medium H plus 2%
ball-milied Wall Street Journal.
-36-
-------
Table XIV
The Synthesis of Protein and Cellulose
by Fungus Cultures Grown on Glucose
in Reese's Medium
Flask No. Culture Temp. Term. pH Protein, rag/ml Cellulose,
mg/ml
1
2
3
4
5
P9
P10
P26
C-264
Control
30
30
30
45
30
2.4
2.4
2.4
2.3
5.1
0.93
1.13
0.75
0.42
_
0
0
2.7
1.4
0
Inoculum; 2 day old culture in Medium R with 10% glucose
Harvest flask Medium: Medium R with 107o glucose, 5 days' inc.
-37-
-------
Table XV
Protein Yields from Newspaper Chopped Dry
in a Waring Blendor Compared to Ball-
Milled Newspaper in Medium H
Flask No. Culture Temp.
1 P9 30
2 P9 30
3 C264 45
4 C264 45
5 P9 . 30
6 C264 45
7 Control (none) 30
Term. pH
3.4
3.4
3.4
3.4
3.3
3.5
4.8
Protein, mg/ml
0.14
0.14
0.15
0.17
0.36
0.25
Inoculum: 2-day culture in Medium R with 2% ball-milled newspaper
(Wall Street Journal)
Harvest-Flask Media; Medium H with 2% ball-milled newspaper
(Wall Street Journal) in flasks 5 and 6. Medium H
with 2% newspaper chopped in Waring blendor in
flasks 1-4.
-38-
-------
Table XVI
Flask No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
The Effect of
Elements
Culture
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
pH Calcium Carbonate Buffering and
on Cellulose Utilization by Fungi
in Medium G
Temp. Term. pH Trace Elements
30
30
30
37
37
30
30
30
37
37
30
30
30
37
37
30
30
30
37
37
30
30
30
37
37
30
30
30
37
37
Control (none) 37
it it
37
3.3
3.3
3.1
3.9
3.4
3.1
3.2
2.9
3.7
3.8
5.0
5.0
5.2
5.2
5.3
5.0
4.9
5.1
5.3
5.4
5.6
5.5
5.6
5.8
5.9
5.8
5.8
5.6
5.7
5.8
4.8
4.9
Yes
ii
ii
ii
ti
No
ti
ii
ii
ii
Yes
ii
ii
11
ii
No
ii
ii
ii
ii
Yes
ii
ii
ii
11
No
ii
n
ii
n
Yes
No
Trace
pH Control
No
n
n
n
n
n
ii
n
n
n
CaC03
n
n
n
n
n
n
n
11
n
Titr.
n
n
n
n
n
n
n
n
n
No
No
% Cellulose
Consumed
5
24
35
15
37
44
35
48
35
31
44
44
58
41
58
60
58
30
58
62
73
62
42
72
67
74
74
66
67
63
0
0
-39-
-------
Table XVI (cont)
The Effect of pH Calcium Carbonate Buffering and Trace
Elements on Cellulose Utilization by Fungi
in Medium G
Flask No. Culture
Temp. Term. pH Trace Elements pH Control % Cellulose
Consumed
33
34
Control(none) 37
ii ii 37
5.2
5.3
Yes
No
CaC03
CaCO,
0
0
Inoculum; 3-day culture in Medium R with 2% ball-milled newspaper
(Wall Street Journal)
Harvest flasks: Medium G with 0.005% bromcresol green indicator
and 2% ball-milled newspaper. (Wall Street Journal).
Harvested at 7 days.
The pH value was maintained in the range 5-6 on
flasks 21-30 by daily titration with sterile 0.1 N NaOH.
(labelled "titr". in table) In flasks 11-20 pH control
was achieved by incorporating 1% OaCO, in the medium.
-40-
-------
Table XVII
Flask
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
The Effect of Trace Elements, Proteose Peptone and Urea
on Protein Yield and Cellulose Utilization
by Fungi Growing on Newspaper in Medium G
No. Inoculum Culture Temp. Nutrient Term.pH Protein
Med. Supplement mg/ml
R
II
II
11
II
G
II
11
II
II
G + trace
"
it
-
-
G + trace
+ Proteose
n
n
11
n
R
II
11
II
rr
G
n
ti
n
n
G + trace
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
els. P9
P10
P26
353x85-1
C264
els.
peptone P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
els. P9
30 None
30
30 "
37
37 "
30 "
30 "
30 "
37 "
37
30 "
30 "
30 "
37 "
37
30 "
30 "
30 "
37 "
37 "
30 Trace els.
30 " "
30 " "
37 " "
37 " "
30 " "
30 " "
30 " "
37 " "
37 " "
30 " "
3.4
3.4
4.2
3.5
3.7
4.5
4.8
3.6
3.6
3.5
4.8
4.2
4.0
3.6
3.5
3.6
3.6
4.2
3.6
3.7
3.5
3.6
5.0
3.7
3.9
5.2
5.2
4.2
4.0
3.7
5.3
0.30
0.30
0.06
0.22
0.26
0.06
0.02
0.14
0.18
0.26
0.02
0.02
0.06
0.22
0.26
0.18
0.26
0.06
0.14
0.26
0.39
0.39
-0.03
0.29
0.35
0.05
0.05
0.09
0.29
0.29
-0.03
% Cellulose
Utilized
19
25
22
25
25
19
19
24
24
35
42
34
28
-41-
-------
Table XVII (cont)
The Effect of Trace Elements, Proteose Peptone and
on Protein Yield and Cellulose Utilization
by Fungi Growing on Newspaper in Medium G
Flask No. Inoculum Culture Temp. '" Nutrient Term.pH
Med . Supp lement
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
G+trace els. PlO
" P26
353x85-1
" C264
G+trace els.
+proteose peptone P9
PlO
P26
" 353x85-1
11 C264
R P9
" PlO
P26
I ,
" 353x85-1
" C264
G P9
" PlO
P26
" 353x85-1
" C264
G + trace els. P9
11 PlO
11 P26
11 353x85-1
" C264
G + trace els.
+ proteoSe peptone P9
" PlO
P26
" 353x85-1
C264
R P9
30
30
37
37
30
30
30
37
37
30
30
30
37
37
30
30
30
37
37
30
30
30
37
37
30
30
30
37
37
30
Trace els. 5.1
" " 4.8
" " 4.0
11 " 3.8
" " 3.9
" " 4.0
" " 4.8
" " 4.0
" " 3.8
trace els. and
proteose peptone 3.5
3.4
3.9
3.9
3.9
" 3.5
3.4
3.6
3.8
" 3.7
" 4.0
3.4
" 3.0
3.9
3.8
3.3
3.3
4.2
" 3.8
3.8
Trace els, pro- 5.8
teose peptone &
urea
Urea
Protein
mg/ml
0
0
0.18
0.25
0.18
0.18
0.03
0.25
0.25
0.26
0.30
-0.12
0.14
0.22
0.11
0.30
0.14
0.18
0.22
0.11
0.30
-0.04
0.11
0.18
0.26
0.30
0
0.18
0.18
0.29
% Cellulose
Utilized
35
35
35
lost
23
26
23
23
17
26
35
50
-42-
-------
Flask No.
Table XVII (cont)
The Effect of Trace Elements, Proteose Peptone and Urea
on Protein Yield and Cellulose Utilization
by Fungi Growing on Newspaper in Medium G
Inoculum Culture Temp. Nutrient Term.pH
Med.
Nutrient
Supplement
Protein
mg/ml
7. Cellulose
Utilized
62
80
81
82
83
84
P10
C264
Control (none)
30
63 "
64
65 "
66 G
67 "
68 "
69 "
70 "
71 G+trace els.
72
73 "
74 "
75 "
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
76 G+trace els.
+proteose peptone P9
77
78 "
79
P10
P26
353x85-1
30
37
37
30
30
30
37
37
30
30
30
37
37
30
30
30
37
37
30
30
30
30
Trace els., pro-
teose peptone,
and urea
5.7
Trace els. ,pro-
teose peptone
& urea
5.3
4.8
5.2
0.26
None
Trace -els.
Trace els. & pro-
teose peptone 4.8
Trace els.,pro-
teose peptone & urea 5.0
0.62
0
0
lost
5.9
5.6
5.4
4.5
4.5
5.8
5.8
5.6
4.4
4.6
5.6
5.3
5.3
4.9
5.0
5.0
5.4
-0.08
0.84
0.55
0.32
0.32
-0.04
0.84
0.56
0.44
0.44
0
0.40
0.44
0.40
-0.04
-0.04
0.44
59
61
39
52
59
67
43
50
65
70
59
70
61
0
0
Inoculum; 2-day cultures in medium R, medium G, medium G supplemented with
"trace elements for medium G" (see Appendix), trace elements plus
0.1% proteose peptone, or trace elements, 0.17. proteose peptone and
0.037. urea, and 27. ball-milled newspaper (Wall St. Journal).
Harvest flasks; Medium G, supplemented with "trace elements for medium G,"
trace elements plus 0.1% proteose peptone, or trace elements plus 0.1%
proteose peptone plus 0.037. urea, and 27. ball-milled newspaper
(Wall St. Journal). Harvested in 5 days.
-43-
-------
Table XVIII
Flask
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Further Studies on the Effect of Trace Elements Protopeptone
and Urea on Protein Yields from Fungi Growing in Medium
G and Reese's Medium
I. Inoculum Cultures
No. Culture Medium Temp. ,°C Term. pH Protein mg/ml
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
R
n
n
ii
n
G
,i
"
„
,,
G + urea
n
n
ii
n
G + urea
+ trace els.
„
n
ii
n
30
I!
II
37
37
30
30
30
37
37
30
30
30
37
37
30
30
30
37
37
P9 G + urea +
trace els. 30
+ proteose peptone
P10
P26
353x85-1
C264
None (control)
„
II II Q
11 II Q
11
M
n
n
R
G
+ urea
+ urea
trace els.
" + proteose
30
30
37
37
30
30
30
30
peptone
3.3
3.3
4.0
5.9
4.3
4.5
4.5
3.8
3.6
3.8
4.6
4.6
4.4
7.3
6.7
4.5
4.5
4.3
7.2
6.5
4.3
4.7
3.9
7.3
6.5
4.9
4.6
4.6
4.6
30 4.6
0.50
0.34
0.14
0.16
0.34
0
0
0
0.04
0.04
-0.04
-0.04
0
0.08
0.08
-0.04
-0.04
0
0.08
0.08
0
0
0.12
0.08
0.20
-
-
-
-
.44-
-------
Table XVIII (cont)
Further Studies on the Effect of Trace Elements Protopeptone
Flask No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
and Urea on
Protein Yields from
6 and
II.
Fungi
Growing in
Medium
Reese's Medium
Harvest Cultures
(Inoculated with above Inoculum Cultures)
Inoculum Culture Medium Temp. , C Term.
Culture No. pH
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
1
2
3
4
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
G
11
it
ii
ii
ii
ii
ii
n
ii
n
ii
ii
n
G
n
n
n
n
n
n
"
M
n
11
30
30
30
37
37
30
30
30
37
37
30
30
30
37
37
30
30
30
37
37
30
30
30
37
37
3.0
3.0
2.8
3.3
3.3
3.1
3.0
2.8
3.8
3.3
3.1
3.1
2.9
3.2
3.3
3.0
3.0
2.9
3.2
3.3
3.1
3.1
2.8
3.2
3.3
G+trace els. 30 3.1
•fproteose peptone
n
n
n
30
30
37
3.1
3.3
3.4
Protein % Cellulose
mg/ml Consumed
0.16
0.20 13
0.30
0.12
0.12
0.12
0.12
0.22
0.02
0.12
0.20
0.16
0.30
0.16
0.12
0.20
0.20
0.26
0.12
0.12
0.22 '
0.22
0.33
0.16
0.16
0.30
0.34
0.41
0.16
-45-
-------
Table XVIII (cont)
Further Studies on the Effect of Trace Elements Protopeptone
and Urea on Protein Yields from Fungi Growing in Medium
G and Reese's Medium
II. Harvest Cultures
Flask No.
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(Inoculated with above
Inoculum Culture Medium
Culture No.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
1
2
3
4
5
6
7
8
9
10
Inoculum Cultures)
Temp. ,°C Term.
pH
C264 G+trace els. 37
P9 -fprofieose peptone
P10 "
P26 "
353x85-1 "
C264 "
P9 "
P10 "
P26 "
353x85-1 "
C264
P9 "
P10 "
P26
353x85-1 "
C264 "
P9 "
P10 "
P26 "
353x85-1 "
C264 "
P9 G+urea
P10 "
P26
353x85-1 "
C264
P9 "
P10 "
P26 "
353x85-1
C264 "
30
30
37
37
30
30
30
37
37
30
30
30
37
37
30
30
30
37
37
30
30
30
37
37
30
30
30
37
37
3.6
3.1
3.0
3.0
4.3
3.5
3.2
3.1
3.0
3.4
3.6
3.2
3.4
3.0
3.4
3.5
3.2
3.1
3.0
3.5
3.6
4.0
4.9
4.6
7.2
6.7
4.3
3.3
4.7
5.0
6.6
Protein
mg/ml
0.16
0.30
0.24
0.22
0.01
0.16
0.34
0.30
0.34
0.22
0.16
0.24
0.30
0.30
0.24
0.18
0.34
0.34
0.41
0.24
0.18
0.44
0.60
0.50
0.27
0.60
0.42
0.16
0.38
0.38
0.64
% Cellulose
Consumed
66
19
62
-46-
-------
Table XVIII (cont)
Further Studies on the Effect of Trace Elements Protopeptone
and Urea on Protein Yields from Fungi Growing in Medium
G and Reese's Medium
II. Harvest Cultures
(Inoculated with above Inoculum Cultures)
Flask No.
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
Inoculum
Culture No.
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Culture
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
Medium Temp. , C
G+urea 30
" 30
11 30
n 37
n 37
11 30
11 30
" 30
" 37
11 37
" .30
30
" 30
11 37
n 37
G+trace els.
+ urea 30
11 30
30
n 37
n 37
" 30
" 30
" 30
" 37
n 37
" 30
" 30
" 30
11 37
n 37
Term.
pH
4.3
3.5
7.0
7.1
4.7
4.2
4.2
3.9
4.8
7.2
5.0
4.4
3.8
3.9
6.3
5.1
3.8
3.9
5.0
6.0
4.2
3.7
3.6
4.9
4.8
3.0
3.3
4.6
6.2
5.1
Protein
rag /ml
0.45
0.24
0.34
-
0.50
0.50
0.34
0.38
0.38
0.38
0.45
0.48
0.34
0.42
0.64
0.48
0.48
0.34
0.34
0.48
0.45
0.16
0.34
0.38
0.50
0.45
0.20
0.24
0.30
0.48
% Cellulose
Consumed
62
4
-47-
-------
Table XVIII (cont)
Further Studies on the Effect of Trace Elements
and Urea on Protein Yields from Fungi Growing
G and Reese's Medium
II. Harvest Cultures
Protopeptone
in Medium
(Inoculated with above Inoculum Cultures)
Flask No.
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
Inoculum
Culture No.
16
17
18
19
20
21
22
23
24
25
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Culture
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
353x85-1
C264
P9
P10
P26
Medium Temp. ,°C
G+trace els 30
+ urea
" 30
" 30
37
37
30
30
30
n 37
37
G+trace els
+proteose peptone
+ urea 30
30
30
n 37
n 37
30
30
30
ii 37
" 37
" 30
30
" 30
37
37
30
" 30
" 30
Term.
PH
4.3
3.7
5.0
4.8
4.9
4.7
4.2
4.6
4.8
4.7
7.0
5.1
6.4
6.1
6.4
7.0
3.7
3.3
7.0
6.2
4.2
4.0
4.2
5.2
6.2
6.8
4.8
4.8
Protein 7. Cellulose
mg/ml Consumed
0.45
0.27
0.2/
0.34
0.45
0.42
0.45
0.38
0.38
0.42
0.40
0.48
0.24
0.24
0.34
0.38
0.38
0.70 42
0.05
0.34
0.38
0.38
0.16
0.27
0.40
0.34
0.34
0.24
-48-
-------
Table XVIII (cont)
Further Studies on the Effect of Trace Elements Protopeptone
and Urea on Protein Yields from Fungi Growing in Medium
G and Reese's Medium
II. Harvest Cultures
(Inoculated with above Inoculum Cultures)
Flask No.
Inoculum
Culture No.
Culture Medium
Temp. , C Term.
PH
Protein
mg/ml
% Cellulose
Consumed
119
19
353x85-1
G+trace els. 37
+proteose pep-
tone + urea
6.6
0.27
120
121
122
123
124
125
126
127
20
21
22
23
24
25
Control
it
C264
P9
P10
P26
353x85-1
C264
None
None
128
129
130
n
n
G+trace els. 30
+proteose
peptone
G+urea 30
G+trace els
+urea 30
G+trace els
+proteose pep-
tone +urea 30
37
30
30
30
37
37
30
30
5.4
7.3
7.0
6.3
7.3
7.5
4.6
4.8
0.38
0.20
0.34
0.24
0.00
0.16
-
_
4.6
4.6
0
0
Inoculum; 2-day cultures in medium R or medium G supplemented as in Part I
of the Table with 0.03% urea, trace elements, 0.037. urea and trace
elements, trace elements and 0.1% proteose peptone or 0.03% urea
trace elements and 0.17= proteose peptone and 27° ball-milled news-
paper (Wall St. Journal).
Harvest Flasks; Medium G supplemented as above plus 27. ball-milled newspaper,
(Wall St. Journal).
-49-
-------
Table XIX
Flask
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
The Comparison of Casamino Acids with Protopeptone as
Nutrient Supplements in Reese's Medium and Medium G
No. Inoculum Culture Medium Term. Protein,
Grown in pH ing /ml
Medium *
RC
ii
ii
Control
RC
M
II
Control
RC
ii
n
Control
RC
M
n
Control
RC
n
n
Control
RC
RC
n
Control
RC
n
ii
P9 R
P10 R
P26 "
None "
P9 RC
P10 "
P26
None "
3.3
3.3
3.3
5.0
3.7
3.6
4.3
5.0
P9 Med G+trace els. 3.0
P10 "
P26 "
None "
P9 Med G+trace
els.+ urea
P10 "
P26
None "
P9 Med G+trace
els.4CaC03
P10 "
P26 "
None "
P9 Med G+trace
els. -(Casamino
acids
P10 Med G+trace
els.+Casamino
acids
P26 "
None "
P9 Med G+trace
els.+Casamino
acids +urea
P10 "
P26
3.1
2.8
4.9
6.8
6.8
3.8
4.9
7.7
7.1
6.7
6.5
3.5
3.5
3.0
4.8
7.3
7.4
7.4
0.42
0.38
0.28
-
0.58
0.48
0.44
-
0.18
0.14
0.26
-
0.62
0.62
0.44
-
0.04
0.04
0.36
-
0.40
0.36
0.32
-
0.34
0.34
0.02
% Cellulose
Consumed
46
43
43
-
46
36
-50-
-------
Table XIX (cont)
The Comparison of Casamino acids with Protopeptone as
Nutrient Supplements in Reese's Medium and Medium G
Flask No. Inoculum Culture Medium Term. Protein, % Cellulose
Grown in pH mg/ml Consumed
Medium *
28
29
30
31
32
Control
RC
Control
None
P9
P10
P26
None
Med.G+trace 4.9
els.+urea +
Casamino acids
Med.G+trace 5.6
els.+urea +
proteose peptone
11 5.5
" 7.3
" 4.9
0.68
0.64
0.06
51
55
19
Inoculum; 2 day cultures in medium R or RC
Harvest Flasks: Medium R or RC, or Medium G supplemented with trace
elements, trace elements plus 0.03% urea, trace elements +
17. CaCO- , trace elements + 0.1% casamino acids, trace
elements + 0.03% urea + 0.1 % casamino acids, or trace
elements + 0.03% urea + 0.1 % proteose peptone.
Harvested in 5 days.
-51-
-------
Table XX
The Influence of the Ingredients of Reese's
on Growth and
Flask No. Culture
Flask
1
2
3
4
5
6
7
8
9
10
11
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Inoc,
1
2
3
None
1
2
3
None
1
2
3
P9
P10
P26
Control
P9
P10
P26
Control
P9
P10
P26
Control
P9
P10
P26
Control
Culture .
Dist.Tap
H20 H20
P9 x
P10 x
P26 x
x
P9 x
P10 x
P26 x
x
P9 x
P10 x
P26 x
Protein Yields of Fungi Growing on
I. Inoculum Cultures
Medium
Newspaper
Medium Term. pH Protein, mg/ml
RC 3.3
11 4.3
" 4.9
" 4.7
RC , using
tap water 4.3
" 3.4
" 5.9
" 4.9
RY 3.9
" 3.3
" 4.9
" 4.8
RG 3.8
3.7
" 5.6
" 4.8
II. Harvest Cultures
Media
KH,PO, (NH,),SO, Urea MgSO,- CaCl.
1 * * l * 7 H29 2
X X X X X
X X XXX
X X XXX
X X XXX
X X XXX
X X XXX
X X XXX
X X XXX
X X XXX
X X XXX
X X XXX
0.24
0.30
0.16
-
0.33
0.51
0.15
-
0.43
0.43
0.19
-
0.26
0.38
0.08
-
, CA Trace Term. Protein
Els. pH mg/ml
x x 3.4 0.58
x x 3.5 0.50
x x 4.2 0.32
x x 4.7 -
x x 3.9 0.56
x x 3.7 0.63
x x 5.4 0.52
x x 4.9 -
x 3.0 0.50
x 5.0 0.00
x 4.1 0.58
7. Cellu-
lose
Consumed
61
62
69
-
50
54
63
-
-52-
-------
Table XX (cent)
Flask
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
Inoc.
None
1
2
3
None
1
2
3
None
1
2
3
None
1
2
3
None
1
2
3
None
1
2
3
None
1
2
3
None
1
2
3
None
Culture
Dist.Tap
-
P9
?10
P26
-
P9
P10
P26
-
P9
P10
P26
-
P9
P10
P26
-
P9
P10
P26
-
P9
P10
P26
-
P9
P10
P26
-
P9
P10
P26
.
X
X
X
X
'• x
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
II. Harvest Cultures
Media
KH.PO. (NH,).SO, Urea MgSO, • CaCl, CA Trace
* * * 7 H20 Els.
X
X
X
X
X
X
X
X
X
X
X
X
X
, X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
XXX
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
XXX
XXX
XXX
XXX
X X ' X
XXX
XXX
XXX
X X
X X
X X
X X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Term. Protein
pH rag /ml
5.0
3.2
4.8
3.4
4.8
4.2
3.8
5.2
4.9
4.2
4.0
6.3
5.0
2.9
5.0
2.6
4.9
6.3
5.2
6.8
5.1
3.6
3.6
3.5
4.7
3.5
3.2
3.2
5.0
2.8
2.8
2.5
5.0
-
0.42
0.00
0.36
-
0.65
0.58
0.50
-
0.65
0.65
0.50
-
0.43
0.00
0.54
-
0.50
0.47
0.28
-
0.48
0.58
0.48
-
0.70
0.85
0.85
-
0.47
0.32
0.72
-
%Cellulosi
Consumed
50
60
44
51
60
74
71
74
-53-
-------
Table XX (cont)
Flask
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Inoc.
1
2
3
None
1
2
3
None
1
2
3
None
1
2
3
None
II. Harvest Cultures
Culture Media
Dist.Tap KH.PO. (NH,)_SO, Urea MgSO,- CaCl. CA Trace Term.Protein ^Cellulose
H20 H20 7 H20 Els. pH mg/ml Consumed
P9
P10
P26
-
P9
P10
P26
-
P9
P10
P26
-
P9
P10
P26
.
XX X
XX X
XX X
XX X
X X
X X
X X
X X
XX X XXX
XX X XXX
XX X XXX
XX X XXX
XX X X
X , X X X
XX X X
XX X X
4.7
4.7
4.9
5.0
4.3
4.3
8.0
4.9
3.2
3.3
3.3
4.9
2.8
2.9
2.6
4.8
0.14
0.10
0.10
-
0.07
0.18
0.02
-
0.62
0.36
0.322
-
0.23
0.16
0.30
_
Inoculum; 2 day cultures in medium RC plus 2% ball-milled Wall Street Journal
Harvest Flasks; Medium R with 2% ball-milled newspaper (Wall St. Journal) supplemented
according to Part II of the Table. Harvested in 5 days.
-54-
-------
Table XXI
Growth and Protein Yields from Fungi Growing
Wall Street Journal vs. Ball Milled Denver
Flask No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Inoculum:
Inoculum
Grown in
RC-4WSJ
u
ii
it
ii
n
RG-fDenver
u
n
u
n
n
Control
Control
on Ball-Milled
Post in
Medium G 8
Culture Paper Term.pH Protein ing /ml
P9
P9
P10
P10
P26
P26
Post P9
P9
P10
P10
P26
P26
None
None
WSJ
n
n
n
u
n
DP
n
n
u
ti
n
WSJ
DP
2 day cultures in medium RC
Wall Street Journal or 2%
6.7
4.0
5.0
4.9
3.6
3.5
5.0
4.3
6.0
4.1
3.9
4.1
4.5
4.7
0.54
0.59
0.63
0.59
0.52
0.48
0.74
0.67
0.63
0.63
0.45
0.41
-
-
% Cellulose
Consumed
67
48
60
67
58
61
61
50
63
53
46
46
0
0
and either 2% ball-milled
ball-milled Denver Post.
Harvest Flasks: Medium G8 and either 2% ball-milled
Journal
or 27o ball-milled Denver Post.
Wall Street
Harvested
in 5 days.
-55-
-------
Table XXII
Growth and Protein Yields from Fungi Grown on
Ball-Milled Wall Street Journal Heated at
200°C for 25 Minutes vs. Unheated in Med.G-8
Flask No.
1
2
3
4
5
6
7
8
Inoculum Culture Paper Term.pH
Medium
RC P9 Unheated
ii plo ii
ii P26 ••
" • None "
" P9 Heated
" P10 "
1, P26
" None "
7.3
5.7
7.6
4.5
4.3
4.4
3.4
4.1
Protein
mg/ml
0.59
0.63
0.13
-
0.68
0.68
0.62
_
% Cellulose
Consumed
57
65
13
-
68
75
62
m.
Inoculum: 2 day cultures in medium RC and 27° ball-mi lied unheated
Wall Street Journal.
Harvest Flasks; Medium G-8 and either heated or unheated 2%
ball-milied Wall Street Journal. Harvested in 5 days.
-56-
-------
Table XXIII
Two-Day Protein Yields from Fungus Cultures Grown on Reese's
Medium Supplemented with Disodium Ethylenediamine tetraacetic
Acid in Place of Protopeptone and 2% Ball-milled Newspaper
Flask No. Culture Medium
Term. pH
Protein, mg/ml
1
2
3
4
5
6
7
8
9
10
11
12
Inoculum:
P9
P10
P26
Control
P9
P10
P26
Control
P9
P10
P26
Control
1 ml of a
R
n
ii
n
R+0.
less
n
ii
ii
R+0.
less
n
n
ii
3.1
2.9
3.1
4.7
1% EDTA 3,4
protopeptone
3.3
4.0
4.9
01% EDTA 2.9
protopeptone
2,9
3.1
4.7
suspension from an agar
0.58
0.54
0.25
-
0.16
0.16
0.06
•
0.09
0.07
0.04
-
slant culture in 5 ml
of distilled water added to each fermentation flask.
-57-
-------
Table XXIV
Comparative Yields of Protein and Cellulose Utilization in Five
Days from Reese's Medium and Medium G Modified by Omitting Ingredients
and Substituting Other Ingredients
o
&
CO
ca
1-1
p*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Culture
P9
u
P10
II
P26
»
-
P9
II
P10
"
P26
M
-
P9
M
P10
tf
P26
"
-
P9
11
P10
II
P26
"
-
P9
II
P10
Distilled
Water
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
K
CU
u
5
D.
CO
H
X
X
X
X
X
X
X
X
X
X
X
X
X
X
£
£
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
0*
co
CM
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
at O
Ld **rt
Ew) r**
S 0*
O CO
0 If
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X
X
X
X
X
X
X
X
X
X
X
X
X
X X
X X
X X
co
« CD O 4-1 K O
O rH CO PN Id rH •
B cu cu Si co cu -a
i-f cu B CM >* co
CM B M CU US 3 <~> tJ rH CU g
rH « -O O -A •* ffl O O
eg COO M O CU. 55 CU? £o
O O •< H t-4 S ^ >•" < H "«
XX X
XXX
XXX
XX X
XX X
XX X
XX X
XX X
XXX
XXX
XX X
XXX
XXX
XX X
X
X
X
X
X
X
X
X
X
X
X
X
X
XXX
XXX
XXX
CU
CO
to" -S
Q\ *O
M Q}
Reese
II
»
K
H
H
H
M
M
M
M
H
M
If
II
II
II
II
II
II
II
II
II
II
X "
II
II
II
II
II
II
a
•
(U
H
3.
3.
3.
3.
3.
3.
4.
3.
3.
3.
3.
3.
3.
5.
3.
3.
3.
3.
3.
2.
4.
3.
3.
3.
3.
3.
3.
4.
3.
3.
3.
Protein ,
mg/ml
3 0.54
4 0.54
4 0.56
4 0.56
2 0.60
4 0.56
7 -
4 0.60
5 0.64
4 0.56
6 0.56
5 0.68
7 0.54
0 -
0
3 0.54
4 0.51
5 0.58
2 0.50
9 0.76
7 -
5 0.55
5 0.59
5 0.57
4 0.44
6 0.49
6 0.59
8 -
1 0.45
0 0.50
0 0.54
^Cellulose
Utilized
47
47
45
58
54
56
-
62
48
54
57
57
61
-
-58-
-------
Table XXIV
Comparative Yields of Protein and Cellulose Utilization in Five
Days from Reese's Medium and Medium G Modified by Omitting Ingredients
and Substituting Other Ingredients
o
55
X
at
a
r-l
fH
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
Culture
Distilled
Water
P10 x
P26 x
P26 x
x
P9
It
P10
tl
P26
IT
-
P9 x
" x
P10 x
" x
x
" x
X
P9
P9
P10
11
P26
n
-
P9 x
" x
P10 x
" x
P26 x
11 x
x
rl
01
U
a r-4 CUiS
r-l «T>O -rl -^MOO
cj ra-H
-------
Table XXIV
Comparative Yields of Protein and Cellulose Utilization in Five
Days from Reese's Medium and Medium G Modified by Omitting Ingredients
and Substituting Other Ingredients
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
J
CO
a
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
0) 01
01 i-i U
H r-l CO -*
3 -rl H 3 O
4J 4J 01 &4
r-l 01 *J O. CM
3 i-i • co co a
o o a H 2
P9 x
x
P10 x
11 x
P26 x
x
x
P9 x
" x
P10 x
" x
P26 x
x
x
P9 x
" x
P10 x
" x
P26 x
" x
x
P9 x
11 x
P10 x
" x
P26 x
" x
x
P9 x
11 x
P10 x
" x
CO O
o* 2 =T
en n f*
eg
sc o y^
x
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
CaCl2
Casamino
Acids
x
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
x ,
X
X
X
ID
oi "01 O 4-> « o
l-l 0) Ol CO r-l .
0) 0) S3 01 01 TJ
a) 6 CM !>-, a)
oi et! 3 ^-N *J r-i CDS
O -rl J
X
X
X
X
X
X
X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
(U
01
CO
B-5 03
co
CO -H
4) -o
G
II
11
II
II
II
II
II
II
II
11
II
11
II
It
11
11
II
It
11
11
II
II
II
11
II
II
"
11
II
11
tl
(X
(U
H
5.4
7.2
6.8
6.2
7.5
7.6
4.7
5.9
7.0
7.3
7.4
5.1
7.0
4.6
7.3
7.4
7.3
7.5
6.4
7.0
4.8
3.9
5.4
5.3
4.9
6.6
7.1
4.8
7.4
7.6
7.5
7.5
Protein,
mg/ml
^Cellulose
Utilized
0.78
0.40
0.61
0.72
0.00
0.02
-
0.64
0.50
0.04
0.08
0.04
0.00
-
0.00
0.06
0.00
0.08
0.06
0.00
-
0.58
0.58
0.58
0.64
0.00
0.00
-
0.03
0.03
0.03
0.00
-60-
-------
Table XXIV
Comparative Yields of Protein and Cellulose Utilization in Five
Days from Reese's Medium and Medium G Modified by Omitting Ingredients
and Substituting Other Ingredients
*
*
*
*
*
(0
a
it
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
H4
115
116
117
118
119
120
121
122
123
124
125
126
127
to o
•0 V4 S CO i-l •
Ci ^i Oi £Xj ^Q ^ *^O
•rtj 4> g CM >> 41
CM HO) HJpSa/^ 4J f-l OlS
—1 (8T3O -H N*MOO
U Ol*rf cdM'Opd CO-U (OM
-------
Table XXIV
£
OT
(-1
s.
128
129
130
131
132
133
134
135
136
137
138
139
140
Comparative Yields of Protein and Cellulose- Utilization in Five
Days from Reese's Medium and Medium G Modified by Omitting Ingredients
and Substituting Other Ingredients
wo -* "> <» S
*oj-i w o to co
a> QJ O ^ 33 o r-i to CL, cu ^ • to " 0*0
4) r-l U CO P r- C 0) 0) S3 01 <1) -0 B-! « 33 C ^ Oj
tj --i « -* CN . -3 , a *o °* ""I _, ^ 3
3 -riM 3 O >-s s-2 -I-H
4J U 5) PL,
i-i to 4J a, N
a -H to w pa
u a 3 H S
P9 x
P10 x
" x
P26 x
x
x
P9 x
" x
P10 oc
x
P26 x
x
x
•^ CO O '"' (0 "O w *^t -^'
33 O CO O to-Hn^-OI!
K . op Q re o >J O m Z
^ 0 2 o o«!Hlw2'-'
X
X
X
X
X
X
X
X
X
X
X
X
X
ua o U
CO -u CO )-t
-------
Flask No.
Table XXV
The Influence of Shaking Rate on Protein Yields from Cultures
Growing on Ball-Milled Newspaper in Reese's Medium Supplemented
with Casaminp Acids Instead of Protopeptone (RC).
Protein mg/ml
Culture
Shaking Rate, RPM
PH
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
P9
it
P10
ii
P26
n
Control
P9
ii
P10
n
P26
n
Control
P9
M
P10
n
P26
n
Control
350
n
n
n
n
n
n
175
M
n
n
n
ii
n
264
n
ii
n
n
n
n
3.1
4.0
3.9
4.1
3.5
3.5
4.8
4.2
3.9
4.1
4.5
3.8
3.7
4.8
4.3
4.2
3.8
4.0
3.4
3.3
4.8
lost
0.45
0.45
0.52
0.48
0.45
-
0.57
0.54
0.50
0.54
0.54
0.54
-
0.56
0.52
0.59
0.52
0.59
0.62
_
Inoculum: 2 day cultures in medium RC with 2% ball-milled newspaper
(Benver Post).
Harvest; 5 day cultures in the same medium.
-63-
-------
Flask No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Inoculum:
Table XXVI
The Influence of Paper and Nitrogen Concentration on
Protein Yields from Fungi Growing in Modified Medium G
Culture Medium Paper, Term. pH Protein
Supplement g/100 ml mg/ml
P9
it
M
ii
P10
ii
n
ii
P26
n
n
ii
Control
n
M
n
P9
n
n
n
P10
n
P10
II
P26
n
n
ii
Control
n
n
ti
None
n
n
n
n
ii
ii
n
n
n
n
n
n
n
n
n
lOxthe usual
Nitrogen
n
n
n
M
n
n
n
ii
ii
n
n
tt
n
n
ti
2 -day cultures in medium
2
4
8
16
2
4
8
16
2
4
8
16
2
4
8
16
2
4
8
16
2
4
8
16
2
4
8
16
2
4
8
16
RC with 2%
3.5
3.0
2.8
2.8
3.5
i.9
2.8
2.8
4.0
2.7
2.8
2.9
5.0
4.8
4.6
4.4
3.7
3.2
3.0
2.8
3.7
3.1
3.0
2.8
4.0
3.2
2.8
2.8
4.9
4.8
4.5
4.4
ball -mi lied
0.28
0.55
0.68
0.48
0.45
0.61
0.68
0.68
0.39
0.78
0.78
0.58
-
-
-
-
0.58
0.79
0.79
1.32
0.32
0.52
0.50
1.24
-
0.67
0.91
1.34
-
-
-
.
newspaper (Denver Post
Harvest: 5-day cultures in Medium RY plus different amts. of ball-milled "
-64-
-------
Table XXVII
The Influence of Paper and Nitrogen Concentration on
Protein Yields from Fungi in Modified Medium G
Flask No, Culture Paper (NH.KHPO, Urea Nutrient
g/100 ml . g/100 ml Supplement
g/iiA) mi
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
. 17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
P9 2
4
8
" 16
n 2
" 4
" 8
11 16
n 2
4
11 8
" 16
Control 2
4
" 8
" 16
n 2
" 4
" 8
16
11 2
n 4
Control 8
16
P9 2
P9 4
11 8
" 16
" 2
" 4
" 8
" 16
1 0.03 Cas. acids
ii n n
n ii M
n n n
" " Yeast
Autolysate
M n it
n M ii
n n n
" " None
n n M
it M ti
n n n
11 " Cas. acids
ti n n
n M it
n n it
" " Yeast
Autolysate
n ii n
n n >i
n n M
" " None
n n ii
1 0.03 None
n n ii
5 " Cas. acids
ii 11 ii
11 II H
n n ii
|| " Yeast
Autolysate
n ii ••
n ii n
n ii "
PH
6.7
7.0
3.5
3.0
6.9
4.0
3.2
2.7
6.8
3.9
3.2
2.9
4.8
4.5
4.3
4.2
4.7
4.5
4.3
4.2
4.5
4.3
4.3
4.1
6.4
4.1
6.2
5.6
5.5
3.8
3.3
4.2
Prote in
ing /ml
0.02
0.60
1.30
1.94
0.58
0.85
0.89
1.34
0.43
0.67
0.88
1.16
-
-
-
-
-
-
-
-
-
-
-
-
0.30
0.43
0.43
0.02
0.62
0.92
0.52
0.26
-65-
-------
Table XXVII (cont)
The Influence of Paper and Nitrogen Concentration on
Protein Yields from Fungi in Modified Medium G
Flask
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
No. Culture
P9
n
n
n
Control
ii
n
n
n
n
n
n
n
n
n
n
P10
"
n
n
n
n
"
II
"
"
II
11
P26
n
n
n
n
M
Paper
g/100 ml
2
4
8
16
2
4
8
16
2
4
8
16
2
4
8
16
2
4
8
16
2
4
8
16
2
4
8
16
2
4
8
16
2
4
(NH4)2HP04
R/100 ml
5
"
n
"
n
n
M
n
11
n
"
"
n
n
n
n
1
n
"
ft
tt
It
11
"
II
If
"
n
"
n
11
n
11
n
Urea
g/lOOml
0.03
"
n
n
n
n
n
n
n
"
"
ii
n
"
M
II
0.03
ii
11
n
n
M
n
n
n
n
n
n
n
n
n
n
n
n
Nutrient
Supplement
None
n
ii
n
Cas. acids
n
n
n
Yeast
Autolysate
n
ii
n
None
n
M
n
Cas. acids
n
n
n
Yeast
Autolysate
n
n
n
None
n
n
n
Cas. acids
n
n
n
Yeast
Autolysate
n
pH
5.6
3.5
3.4
4.1
4.5
4.4
4.3
4.2
4.5
4.3
4.3
4.1
4.3
4.3
4.2
4.1
6.9
6.8
3.4
3.2
7.0
5.2
3.2
2.9
7.0
4.1
3.1
3.1
6.6
5.3
2.6
2.8
3.2
2.7
Protein
mg/ml
0.44
0.17
0.87
0.12
-
-
-
-
-
-
-
-
-
-
-
-
0.48
0.92
1.28
2.08
0.63
1.17
1.20
0.84
0.64
0.81
0.48
0.96
0.14
0.28
1.08
1.28
0.63
0.85
-66-
-------
Tjtble XXVII (cent)
Flask No
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
The Influence of Paper and Nitrogen Concentration on
Protein Yields from Fungi in Modified Medium G
, Culture Paper (NH,)2HPO, Urea Nutrient
g/lOOml g/100 ml g/lOOml Supplement
P26 8
" 16
n 2
•• 4
" 8
" 16
Control 2
M 4
" 8
" 16
2
n 4
11 8
" 16
n 2
4
" 8
16
P10 2
n 4
" 8
» 16
n 2
n 4
" 8
» 16
n 2
M 4
" 8
» 16
P26 2
11 4
1 0.03 Yeast
Autolysate
n n ii
11 " None
n n n
n n n
ii n n
1 0.03 Cas. acids
n n n
ii n n
n n n
" " Yeast
Autolysate
n n n
M . » 'I
n M ii
" " None
n n n
n ii ii
n tt n
5 " Cas. acids
n H n
H M • ii
M n ii
» " Yeast
Autolysate
H •' "
ii ii ii
n M "
" " None
M n "
II II «'
II « 1'
5 0.03 Cas. acids
n n ii
pH
2.7
2.5
2.9
2.9
2.8
2.6
4.7
4.6
4.5
4.3
4.7
4.6
4.4
4.3
4.5
4.4
4.3
4.3
6.3
6.1
6.0
6.0
6.2
3.7
3.9
5.5
5.5
5.7
5.6
4.5
6.6
6.5
Protein
mg/ml
0.96
0.22
0.56
0.66
0.90
1.46
-
-
-
-
-
-
-
-
-
-
-
-
0.32
0.00
0.00
0.22
0.34
0.67
0.21
0.02
0.65
0.58
0.40
0.00
0.51
0.10
-67-
-------
Table XXVII (cont)
Flask No.
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
Inoculum:
Harvest:
The Influence of Paper and Nitrogen Concentration o'n
Protein Yields from Fungi in Modified' Medium G
Culture Paper (NH,)2HPO, Urea Nutrient pH Protein
g/100 ml e/100 ml g/lOOml Supplement mg/ml
P26 8
11 16
" 2
" 4
" 8
11 16
I. 2
" 4
" 8
16
Control 2
" 4
8
11 16
" 2
4
" 8
11 16
11 2
" 4
" 8
16
3 -day cultures
(Denver Post)
14 -day cultures
medium 6-6 plus
(Denver Post)
5 0.03 Cas. acids
ii ii ii
11 " Yeast
Autolysate
ii ii ii
n ii M
n H n
" " None
n n n
n M n
n n n
" " Cas. acids
n n n
n n n
ii n n
" " Yeast
Autolysate
n n n
n ii n
n n n
" " None
n M n
n ' n n
n M n
3.3 0.19
3.0 0.94
,
6.4 0.22
5.9 0.54
3.2 0.63
2.8 1.66
5.7 0.31
4.6 0.28
3.0 0.52
2.6 1.34
4.7
4.5
4.4
4.1
4.4
4.4
4.3
4.2
4.5
4.3
4.2
4.0
medium RY with 2% ball -milled newspaper,
in Medium G, medium G-l, medium G-5
and
different amounts of ball-milled newspaper
-68-
-------
Table XXVIII
The Effect of Tween-80 on Growth and Protein Production
by Fungi Qrowing on Ball-Milled Newspaper
in Modified Reese's Medium & Medium G
Flask No, Culture Medium Tween-80,mg/ml pH Protein, mg/ml
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Inoculum:
Harvest;
P9
tt
ii
ii
ii
ii
ii
ii
P10
ii
ii
ii
ii
11
ii
n
P26
ii
n
n
n
n
n
M
RY
n
n
n
G
it
n
n
RY
n
n
n
G
n
n
n
RY
n
M
M
G
n
n
n
Control RY
M
n
n
n
M
ii
n
2 -day
5 -day
n
n
M
G
n
n
n
0.00
0.01
0.10
0.50
0.00
0.01
0.10
0.50
0.00
0.01
0.10
0.50
0.00
0.01
0.10
0.50
0.00
0.01
0.10
0.50
0.00
0.01
0.10
0.50
0.00
0.01
0.10
0.50
0.00
0.01
0.10
0.50
cultures in Medium RY with
cultures in
newspaper (Denver
medium RY or in
Post) .
5.2
4.2
3.7
3.5
6.5
6.5
4.0
6.8
3.8
3.9
3.6
3.4
5.3
5.6
5.2
-
3.4
3.3
3.3
3.1
3.7
4.0
3.4
3.7
4.6
4.8
4.8
a. 8
4.5
4.5
4.5
4.5
2% ball-milled
medium G , both
0.29
0.44
0.59
0.48
0.65
0.63
0.67
0.28
0.29
0.51
0.66
0.54
0.73
0.67
0.64
-
0.52
0.57
0.66
0.64
0.47
0.50
0.64
0.64
-
-
-
-
-
-
-
-
newspaper (Denver Post)
with 2% ball-milled
-69-
-------
Table XXIX
Protein Yields from Fungi Growing on Coarsely Shredded
Newspaper vs. Ball-Milled Newspaper in Medium G
Supplemented with Extra Nutrients
Flask No. Culture Supplement Paper Harvest Term. pH Protein
time, days ^ mg/ml
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
1
2
3
4
5
6
7
8
P9
P10
P26
P9
P10
P26
P9
P10
P26
Control
11
n
P9
P10
P26
P9
P10
P26
P9
P10
P26
Control
n
M
P9
P10
P26
P9
P10
P26
P9
P10
Urea
n
n
n
n
ii
n
n
M
M
II
II
Urea and
Yeast autol.
> ti
n
n
n
n
n
n
n
M
n
n
Urea
n
n
n
M
n
ii
n
BM
n
n
S
M
n
SH
n
n
BM
S
SH
BM
ii
n
S
n
n
SH
SH
n
BM
S
SH
BM
n
M
S
M
M
SH
n
5
n
M
ii
n
n
ii
n
n
n
n
n
n
M
II
II
II
II
II
5
n
ti
n
n
5
n
M
ii
n
n
ii
n
4.3
3.8
3.3
5.6
5.3
7.3
5,8
5.9
5.0
4.5
4.3
4.4
4.0
3.7
7.3
7.5
7.4
7.0
7.2
7.0
6.2
4.6
4.4
4.4
7.7
5.0
3.1
7.0
4.9
7.7
6.9
7.0
0.56
0.54
0.35
0.23
0.16
0.00
0.20
0.06
0.04
-
-
-
0.65
0.54
0.00
0.19
0.10
0.00
0.07
0.04
0.03
-
-
-
0.32
0.63
0.24
0.22
0.08
0.02
0.08
0.06
-70-
-------
Table XXIX (cont)
Protein Yields from Fungi Growing on Coarsely Shredded
Newspaper vs. Ball-Milled Newspaper in Medium G
Supplemented with Extra Nutrients
flask Np, Culture Supplement Paper Harvest Term. pH
time, days
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
P26
ControJ,
ii
n
P9
P10
P26
P9
P10
P26
P9
P1Q
P26
Control
ii
n
P9
P10
P26
P9
P10
P26
P9
P10
P26
Control
M
n
Urea
n
n
n
Urea + yeagt
auto ly sate
n
it
ii
n
n
n
n
n
n'
M
n
Urea
n
n
n
n
n
M
n
n
n
n
n
P9 Upea + yeast
auto ly sate
P10
P26
P9
n
M
n
SH
BM
S
SH
BM
n
n
S
S
tl
SH
11
SH
BM
S
SH
BM
n
n
S
n
n
SH
n
ii
BM
S
SH
BM
n
n
S
5
ii
n
n
M
n
n
n
n
n
n
n
5
1!
It
II
9
n
n
n
ti
n
n
n
n
11
ii
n
M
n
n
n
4.5
4.5
4.4
4.2
4.1
4.4
7.6
7.6
7.6
7.6
7.4
7.3
5.1
4.6
4.6
4.2
3.1
3.5
6.8
7.2
7-1
7.3
7.0
6.4
5.7
4.4
4.4
4.1
4.8
6.6
2.9
7.3
Protein
rag /ml
0.06
-
-
-
1.00
0.98
0.08
0.04
0.04
0.00
0.05
0.19
0.08
-
-
-
1.31
0.63
0.13
0.00
0.09
0.07
0.02
0.00
0.00
-
-
-
0.58
0.63
0.76
0.09
-71-
-------
Table XXIX (cont)
Flask No
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
+ Paper
Inoculum
Harvest;
Protein Yields from Fungi Growing on Coarsely Shredded
Newspaper vs. Ball-Milled Newspaper in Medium G
Supplemented with Extra Nutrients
Culture Supplement Paper Harvest Term, pH Protein
time, days rag/ml
P10
P26
P9
P10
P26
Control
ii
ii
P9
PlO
P26
P9
PlO
P26
P9
PlO
P26
Control
it
it
P9
PlO
P26
P9
PlO
P26
P9
PlO
P26
Control
n
ii
Urea + yeast
auto ly sate
n
n
n
n
n
n
n
Urea
n
n
n
n
M
II
II
II
II
II
II
Urea + yeast
autolysate
n
n
n
n
n
n
it
n
n
n
n
S
n
SH
n
n
BM
S
SH
BM
n
ti
S
ii
n
SH
ti
n
BM
S
SH
BM
n
n
S
n
n
SH
n
ii
BM
S
SH
- BM=ball-milled , S=shredded ,
: 2-day cultures in medium RY
Media Gl
and G3 harvested
in
9
n
n
n
n
n
n
n
14
n
n
n
n
n
n
n
n
it
n
n
n
n
ii
n
ii
11
11
11
ii
n
it
n
7.2
7.4
7.2
7-1
6.6
4.6
4.5
4.3
6.8
6.6
3.9
6.8
6.9
7.0
6.8
6.7
3.7
4.6
4.5
4.3
6.6
6.9
2.9
6.9
6.6
6.3
6.6
7.1
6.5
4.7
4.6
_
SH=shredded and heated to 200 C
with ball-milled
5 , 9 and 14 days .
newspaper + 2%
0.00
0.00
0.00
0.00
0.55
-
-
-
0.56
0.61
0.51
0.28
0.30
0.15
0.29
0.24
0.53
-
-
-
0.59
0.59
0.67
0.20
0.13
0.17
0.00
0.00
0.16
_
_
_
for 15 min.
Denver Post
-72-
-------
Table XXX
Stirred -Jar Fermentor Studies with Myrothecium verrucaria
Run
No.
3
4
5
6
7
Grown on Newspaper
A
Fermentor Medium Paper Initial Term. Inc. Stirring
No. PH pH time, rate
days RPM
I
2
3
1
2
3
1
2
3
1
2
3
1
2
3
F,10
G,10
H,10
G,10
G,10
G,10
G,10
6,10
G.10
G-l,
G-l,
G-2,
G-3,
G-3,
G-3,
1 BMJ,2%
1 BMW, 2%
1 BMf,2%
1 BMW, 2%
1 BMW ,2%
1 BMW, 2%
1 B!W,2%
1 BMf,2%
1 BMW, 2%
101 BMW, 2%
101BJW,4%
101 BMW, 2%
101 BMD,2%
10 1 BMD ,4%
BMD ,8%
5.3
5.3
5.3
5.3
5.3
5.2
5.3
5.3
6.5
5.3
5.3
5.3
5.3
5.3
5.3
3.251
4.90
4.15
3.45
4.652
6.50
4.00
4.352
6.402
5.50
4.95
5,20
5.75
5.30
5.40
9
9
9
12
12
12
14
14
14
21
21
21
24
24
24
100
100
100
0
0
0
0
0
0
0
0
0
100
100
100
Aeration Inoculum
rate,
1/min
2.4
2.4
2.4
2.4
2.4
2.4
2.4
2.4
2.4
2.4
2.4
2.4
2.4
2.4
2.4
R, 200ml
R ,200ml
R, 200ml
R, 200ml
R, 200ml
R ,200ml
4 -200ml3
4 -200ml
4 -200ml
R, 200ml
R, 200ml
R, 200ml
RY, 200ml
RY, 200ml
RY, 200ml
Protein % Cellu-
yield lose
mg/ml Utilized
0.48
0.51
0.44
0.42
0.47
0.35
0.44
0.48
0.30
0.22
0.57
0.25
0.25
0.34
0.63
46
67
62
59
62
64
58
52
54
63
57
57
77
74
45
6.57 1
8
9
10
11
12
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
G-l,
G-l,
G-3,
G-3,
G-3,
G-3,
G-3,
G-3,
G-3,
G-3,
G-3,
G-3,
G-3,
G-4,
G-l,
10 1 BMD, 2%
10 1 BMW ,2%
10 1 BMD, 2%
10 1 BMD ,4%
10 1 BMD ,4%
10 1 BMD ,4%
10 1 BMD ,4%
10 1 BMD ,4%
10 1 BMD ,4%
10 1 BMD ,4%
10 1 BMD ,4%
10 1 BMD ,4%
7.5 1 BMD ,4%
7.5 1 BMD ,4%
715 1 BMD ,4%
5.3
5.3
5.3
5.3
5.3
5.3
5.3
5.3
5.3
5.3
5.3
5.3
5.3
5.3
5.3
5.65
5.55
5.80
5.65
5.55
5.35
4.50
4.50
4.55
4.15
4.10
3.60
4.05
4.15
4.40
20
20
20
18
18
18
14
14
14
11
11
11
12
12
12
100
100
100
100
100
100
300-4 OO5
300-400
300-400
400
400
400
400
400
400
2.4
2.4
2.4
1.5
3.0
6.0
1.5
3.0
6.0
3.0
3.0
3.0
3.0
3.0
3.0
RY, 200ml
RY, 200ml
RY, 200ml
RY, 200ml
RY ,200ml
RY, 200ml
RY, 100ml
RY , 100ml
RY, 100ml
RY, 200ml
RY ,200ml
RY, 200ml
RY ,150ml
RY, 150ml
RY, 150ml
0.25
0.40
0.36
0.33
0.47
0.94
1.19
1.27
1.36
1.19
0.97
1.02
1.42
1.11
0.89
70
81
77
34
46
61
63
65
63
68
63
61
62
51
52
-73-
-------
Table XXX
Footnotes:
1. pH was adjusted on day 7 to 5.3 by adding sterile 1 N NaOH. Prior to
this point pH values, never having been adjusted, were:
1 3.05
2 3.55
3 3.25
2. These samples were pH-controlled by automatic titration. See Figure 7 .
3. These inocula consisted of 200 ml of culture from run no. 4, refrigerated
until use. Jar 1 was inoculated from jar 1 of run 4, jar 2 from jar 2,
and jar 3 from jar 3.
4. Paper BMW = ball-milied Wall Street Journal
BMD = ball-milled Denver Post
The amount given as % is grams of paper per 100 ml of culture medium.
5. The higher stirring•rate of run 10 resulted in some foaming losses and
build-up of solids on the walls of the fermentor above the normal liquid
level. Antifoam, (Hodag FD-82, diluted to contain 9 g/1) was therefore
used in runs 11 and 12.
-74-
-------
Table XXXI
Analytical Data on the Dried Ground
Harvest from Stirred-Jar 3, Run No. 10
Moisture Content
Ash Content
7. (By Weight)
Harvest
2.4, 2.5
14, 13
Corn
10.6
1.53
Wheat
10.5
1.8
Biuret, Total Protein
Biuret, Soluble Protein
Ammonia Nitrogen
Organic Nitrogen
"Protein11*
Total Kjeldahl Nitrogen
Nitrate Nitrogen
Nitrite Nitrogen
Total Carbohydrates
Soluble Carbohydrates
Cellulose Content
Total Lipids
2.8, 2.9
2.1, 2.0
4.60, 4.50, 4.53
1.01, 1.05
6.31, 6.57 10.3 11.9
5.61, 5.58
< 0.0005, < 0.0005 -
0.0001, 0.0001
. 29, 31
0.99, 0.93
23, 22 2.2** 1.8**
2.2, 2.3 5.0 2.1
* Organic Nitrogen x 6.25
** 1'Fiber" (cellulose + lignin)
-75-
-------
Figure 1. Stirred-Jar Apparatus in Use for Fermentation of
Ball-Milled Newspaper
-76-
-------
0-5-
s-f
I 9-3
P-l
O-l
0-
© -
10
Figure 2. Protein Production, Stirred-Jar Run 3
Figure 3. Cellulose Utilization, Stirred-Jar Run 3
-77-
-------
Figure 4. Variation in pH, Stirred-Jar Run 3
„.(.
04.
1 W
Figure 5. Protein Production, Stirred-Jar Run 4
-78-
-------
0
Figure 6. Cellulose Utilization, Stirred-Jar Run 4
II II
0 - tfo f H
© - (IK C.»iT*«tcf D Af 43
Figure 7. Variation in pH, Stir red-Jar Run 4
-79-
-------
0-f.
0.5
»«•»!
0-3
I"
o
Figure 8. Protein Production, Stirred-Jar Run 6
Figure 9. Cellulose Utilization, Stirred-Jar Run 6
-80-
-------
ru R re TT *5
. f 04yj
Figure 10.. Cellulose Utilization, Stirred-Jar Run 6
Figure 11. Variation in pH, Stirred-Jar Run 6
-81-
-------
Figure 12. Protein Production, Stirred-Jar Run 7
Figure 13. Cellulose Utilization, Stirred-Jar Run 7
-82-
-------
O 2
Figure 14. Protein Production, Stirred-Jar Run 8
i ^"^ C ?
Figure 15. Cellulose Utilization, Stirred-Jar Run 8
-83-
-------
0.7
o.l.
ftn,o jt.es,
MJ/Kl
8-3
o.l
0 ^ *
IU
Figure 16. Protein Production, Stirred-Jar Run 9
Ob
o-t
3 -i
7 ? 1
Figure 17. Protein Production and Cellulose Utilization, Stirred-Jar
Run 11
-84-
-------
Figure 18. Protein Produced, Stirred-Jar Run 12
Figure 19. Cellulose Utilized, Stirred-Jar Run 12
-85-
-------
References
1. Gascoigne, J. A. and M. M. Gascoigne, I960 Biological Degradation
of Cellulose. Butterworths, London.
2. Casey, J. P. 1952, Pulp and Paper, Chemistry and Chemical Technology.
Interscience Publishers, Inc., New York. Vol. I. Pulping and
Papennaking.
3. Feinstein, R. N. 1949, Modification of the Biuret Method of Protein
Determination, Anal. Chem. 21; 534-539.
4. Crampton, E. ¥. and L. A. Maynard 1938. The Relation of Cellulose and
Lignin Content to the Nutritive Value of Animal Feeds. J. Nutrition,
15;383-396.
5. Scott, T. A. Jr. and Melvin, E. H. 1953, Determination of Dextran with
Anthrone, Anal. Chem. 25: 1657-1661.
6. Updegraff, D. M., 1970, The Semi-Micro Determination of Cellulose in
Biological Materials, Analyt. Biochem., In Press.
7. Society of American Bacteriologists 1957. Manual of Microbiological
Methods.
8. Walseth, C. S. 1952. Occurrence of Cellulases in Enzyme Preparations
from Microorganisms., TAPPI 35_: 228-233.
9. Han, Y. W. and Srinivasan, V. R. 1968. Isolation and Characterization
of a Cellulose-Utilizing Bacterium. Applied Microbiol. 16; 1140-1144.
10. Smith, G., 1969 Industrial Mycology, Sixth ed., Edward Arnold, Ltd.,
London.
11. Gray, W. D. 1962 Microbial Protein for the Space Age. Developments in
Industrial Microbiology 3: 63-71.
12. Ghose, T. K., J. Kostick and E. T. Reese, 1968 Enzymatic Saccharificatioi
of Cellulose in Batch and Semi-Continuous Aerated Systems. Paper given
at the A.C.S. Symposium on Cellulases, Sept. , 1968.
13. Reese, E. T. and A. Maguire, 1969 Surfactants as Stimulants of Enzyme
Production by Microorganisms, Applied Microbiol. 17:242-245.
14. Tannenbaum, S. R. and R. I. Mateles, 1968 Single Cell Protein. MIT
Press, Cambridge, Mass.
15. Bode, V. C. Goebell, H., Stahler, E., 1968, Zur Eliminierung von
Trubungsfehlern bei der Eiweissbestimmung mit der Biuret methode.
Z. Rlin. Chem. u. Klin. Biochem. 5: 418-422.
-86-
-------
16. Robinson, H. W. and Hogden, C. G. , 1940 The Biuret Reaction in the
Determination of Serum Proteins. J. Biol. Chem. 135:707-725.
17- Association of Official Agricultural Chemists, Official Methods of
Analysis, A.O.A.C. , Washington, D.C. , Tenth Edition, 1965.
18. American Public Health Association, Standard Methods for the Examination
of Water, Sewage and Industrial Wastes. A.P.H.A. , New York, Tenth
Edition, 1955.
19. D. F. Beltz, 1958, Colorimetric Determination of the Nonmetals,
Interscience Publishers. New York.
20. Reese, E. T. , Siu, R.G.H. and Levinson, H.S. 1950 Biological Degradation
of Soluble Cellulose Derivatives and its Relationship to the Mechanism
of Cellulose Hydrolysis. J. Bact. 59: 485-497.
21. Mandels, M. and E. T. Reese, 1964, Fungal Cellulases and the Microbial
Decomposition of Cellulosic Fabric. Devel. in Industrial Microbiol.
5_:5-20.
22. Wood, T. M. 1968 Cellulolytic Enzyme System of Trichoderma koningi.
Separation of Components Attacking Native Cotton. Biochem. Jour. 109:217-227.
23. Winton., A. L. and K. B. Winton, 1932, The Structure and Composition
of Foods, John Wiley and Sons, New York.
24. Day, W. C., M. J. Pelczar, Jr. and S. Gottlieb, 1949, The Biological
Degradation of Lignin, I. Utilization of Lignin by Fungi, Arch. Biochem.
23:360-369.
25. Ciegler, A. and E. B. Lillehoj , 1968, Mycotoxins Adv. in Applied
Microbiol. 10:155-219.
-87-
-------
Publications Supported by Grant
1. Updegraff, D. M., 1970, The Semi-Micro Determination of Cellulose
in Biological Materials, Analytical Biochem. In Press.
Staffing
1. Dr. John D. Douros, Principal Investigator, June 1, 1967 - July 15, 1968.
50% of time.
2. Dr. David M. Updegraff, Principal Investigator, July 15, 1968 to date.
50% of time.
3. Dr. Thomas D. Nevens, Senior Research Engineer, June 1, 1967 to date.
15% of time.
4. Miss Sandra Hockersmith, Microbiologist Specialist, June 1, 1967 to
Jan. 1, 1969, 50% of time.
5. Miss L. Myra Fuller, B.S. Chemist, Scientist C., June 1, 1967 to
May, 1968. 50% of time.
6. Mrs. Diane Mack, B.S. , Microbiologist, on project as a technician under
Miss Hockersmith, June 1967 to Dec. 1968. Since then in charge of
fermentations microbiology laboratory, until her departure in May 1969.
7. Mr. Larry Griffin, Technician, carried out all of the 14 liter
stirred-jar fermentor work, and most of the chemical analytical
work on the product.
8. Mrs. Jeannette King, B.S., Microbiologist, worked in the fermentation
laboratory during the entire period of the project.
9. Mrs. Sharon Zaun, M.S. Biologist, worked in fermentation laboratory
from May 1968 to August 1969.
-88-
-------
Appendix
Table I
Culture Medin Used for Isolation of
Hydrocarbon-Oxidizing Organisms and Some Cellulose Utilizers
Medium Concentration g/1 distilled HjO
Medium 1
CaCl2'2H20 .15
KC1 -40
LO 2.0
.9
2.0
NaN03 20.0
1 ml of microelements (Salts B)
Salicylic Acid Medium (pH=7)
-5
-2
-05
-05
MgS04
MnCl2'4H20
KH2P04
Raymonds Medium (pH=7)
MgS04'7H20
KH2P04
CaCl, '
MnSO, °'°02
FeS04«7H20 °'005
-89-
-------
Table I (cont.)
Concentration g/1 distilled H20
Ammonium phosphate (Dibasic) 10.0
Potassium phosphate (Dibasic) 5.0
Sodium sulfate 0.5
Salts "B" Microelements #4 10 cc
Microelements Medium 4 (Salts "B")
7H0 40.0
FeS04'7H20 2.0
4H20 2.0
NaCl 2.0
Yeast Hydrocarbon Medium (pH-6. 5) g/liter tap water
Na2HP04'12H20 2.5
KH2PQ4 3.5
MgS04'7H20 1.0
NaCl 0.5
Urea or NH4N03 5.0
Yeast Medium of British Petroleum (pH=4) g/liter distilled H20
(NH4)2HP04 2.0
KC1 1.15
MgS04'7H20 Q.65
ZnS04-H20 0.17
MnS04'10H20 0.045
PeS04'7H20 0.068
Yeast Extract 0.025
PM Medium
Cad2 0.01
FeS04'7H20 0.1
85% H3P04 1.5
XC1 0.5
MgS04 0.5
MnS04 0.11
Na2S04 0.1
NH4C1 0.11
-90-
-------
Table II
Culture Media Used Primarily_for Ccllulolytic Microorganisms
Bushnell and Haas Medium (pH"7.0 to 7.2) Concentration g/1 distilled H20
20 0.2
20 0.02
1.0
1.0
NH4W>3 1,0
FeClj4H20 2 drops
Modified Czapeka Medium (pH»4.0)
3
1
MgS04-7H20 0.5
KC1 0.5
FeS04'7H20 0.05
Carbon Source 20.0
Srinivasan's Medium (pH«»6.3) (SM)
NaCl 6.0
0.5
0. 5
MgS04 °- l
CaCl2 0>1
Yeast Extract li0
tteaac's Medium (pH"5.3) (medium R or TV medium)
KH2P04 2'°
(w )2so4 l-*
Urea °'3
MgSO,-7H 0 °'3
CaC!* °'3
Proteose Peptone lf
Trace Elements X
-91-
-------
Table II front. 1
Trace Elements for Reese Medium Concentration mg/100 ml distilled H20
MnS04'H20 156.0
FeS04'7H20 500.0
167.0
200.0
191 BC1 1 ml.
F Medium (pH-7.8) Concentration g/1 tap H2<>
10.0
5.0
N«2S04 0. 5
Microelements #4 (Salts B) 10.0 ml.
Microelements #4 Salts B Concentration gtt/1 distilled
MgS04'7H20 40.0
2.0
1.5
NsCL 2.0
Medium RG
Reese's medium with glycine, 0.1%, instead of protopeptone
Medium RC
Reese's medium with casamino acids, 0.1%, (Difco Certified) instead of protopeptone.
Medium RY
Reese's medium with yeast autolysate, 0.1%, (Amber Labs, Inc., BYF Series 100)
instead of protopeptone.
Medium F (pH 5.3) Concentration, g/1 tap H20
(NH4)2S04 (fertilizer grade) 10.0
Na2S203 0.01
Medium G (pH 5.3) Concentration, g/1 tap
(NH4)2HP04 (C.P.) 10.0
Na2S203 0.01
Medium H (pH 5.3) Concentration, g/1 tap H20
(NH4)2S04 5.0
5.0
0.01
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Medium G-l
Medium G + 0.03% urea
Medium G-2
Medium G + 1% CaCO,,
Medium G-3
Medium G + 0.03% urea + 0.1% yeast autolysate (Amber Labs, Inc., BYF Series 100)
Medium G-4
Medium G + 0.03% urea + 0,05% yeast autolysate (Amber Labs, Inc., BYT Series 100)
Medium G-5
Medium G + 4% extra (NH4)2HP04
Medium G-6
Medium G-l -1- 4% extra (NH,)2HPO,
Medium G-7
Medium G + 9% extra (NH4)2HP04
Medium G-8
Medium G-l + 1.0 ml/1 trace elements for medium G
Medium G-9
Medium G + 1.0 ml/1 trace elements for medium G
Trace Elements for Medium G
CaCl2 30 g/1
MgS04 3.0 g/1
CuS04'5H20 0.05 g/1
Usevl ml/liter of media plus 1 ml trace elements for Medium R/liter
media.
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Original Biuret Protein 3» 16 Method
1. To 5.0 ml homogenized sample in a test tube add 1.0 ml 5 N NaOH.
Mix well and allow to stand one hour.
2. Add 0.2 ml 20% copper sulfate. Mix well and allow to stand 30 minutes.
3. Filter through Whatman #1 paper and collect filtrate in another test tube.
4. Read absorbance at 530 mU in 12 mm cuvettes versus a reagent blank
of 5.0 ml distilled water carried through the procedure as above, steps 1-3.
5. Compare with a protein standard curve obtained similarly. A dilution
of Versatol (Warner-Chilcott Labs) containing 5.0 mg protein/ml is
a suitable standard. Use 0, 0.5, 1.0 and 2.0 ml standard diluted to
5.0 ml distilled water to correspond to 2.5, 5.0 and 10.0 mg protein/
5 ml sample. Read the result directly from the curve in mg protein/
5 ml sample.
6. 2.5 mg protein in 5 ml will produce an absorbance of about 0.30.
Therefore, it is possible to detect about 0.1 mg protein per ml sample.
The curve is essentially linear through 10 mg protein/5 ml sample.
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A "blank" is run by the same procedure on the freshly inoculated culture
medium which was used in the fermentation in order to obtain the initial
protein concentration of the medium. The final figure for protein produced
was derived by subtracting this value from the protein value of the
fermentation beer.
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Revised Biuret Protein Method 1J>
1. To 5.0 ml sample in a 15 ml centrifuge tube, add 5.0 ml distilled water
and 2.0 ml 5 N NaOH. Mix well by inversion. Let stand one hour.
2. Centrifuge 5 minutes at high speed in clinical type centrifuge.
3. Transfer 6.0 ml supernatant to another 15 ml centrifuge tube.
4. Add 0.2 ml 20% copper sulfate and mix well. Let stand 30 minutes.
5. Centrifuge 5 minutes at high speed.
6. Decant supernatant into a test tube.
7. Read absorbance (A^ at 550 i*J. in 2 cm cells versus distilled water
on a Beckman DK-2 spectrophotometer.
8. Decolorize the protein complex by adding 0.10 ml 30% KCN (caution - use hood)
mixing well, then allowing to stand exactly 5 minutes.
9. Read absorbance (A,,) as above, step 7. Decolorize only 2-3 samples at
one time in order to obtain A_ within 7 minutes.
10. Subtract A.^ from A.^ to obtain A (the absorbance due to the protein
complex only).
11. Compare with a protein standard curve obtained similarly. A dilution of
Versatol (Warner-Chilcott Laboratories ) containing 5.0 rag protein/ml is
a suitable standard. Use 0, 0.5, 1.0 and 1.5 ml standard diluted to
5.0 ml with distilled water to correspond to 2.5, 5.0 and 7.5 mg protein/5 ml
sample. Multiply the result obtained from the standard curve by 2 to correct
for the 1:2 dilution in step 1. This gives mg protein/5 ml sample.
12. 2.5 mg protein in 5.0 ml will produce an absorbance of 0.28-0.31. Therefore,
it is possible to detect about 0.10 mg protein/ml sample. The curve is
linear through 7.5 mg protein/5 ml sample.
It was shown that lengthy fermentations, expecially with high concentrations
of newspaper (4-8%) generated disproportionately high blank values due to extraction
of highly colored lignins, inks, etc. in the paper by NaOH. These could not be
legitimately subtracted with the use of the initial control samples, since the
fermentation may change the amounts of these non-specific colors as well as the
biuret color.
Therefore, this procedure was developed from modifications of existing
methods15 and was adopted for use on the stirred-jar fermentation runs 6-12 where
a higher level of accuracy was desired.
The initial reading (A^ measures both the biuret protein complex and
interfering color and/or turbidity. A2 (after decolorization) measures all
interferences which are then subtracted to obtain the absorbance due to protein
only.
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CM/WE FoA. R£\/I*EI> BIOA£T Merttoo Fort
VO
00
5" ml
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Calculation of Corrected Protein and Cellulose Values
A. Shake-Flasks
To correct for evaporation losses which occurred during autoclaving
and fermentation, all flasks were diluted to their original volume of 100 ml,
then sampled and analyzed for protein and cellulose content.
The protein produced, or cellulose utilized was calculated by
obtaining the difference from uninoculated controls.
Example; Data from Table XXV
Uncorrected Corrected
Protein Protein
Flask Present Produced
No. mg/ml mg/ml
8 0.91 0.57
10 0.84 0.50
12 0.88 0.54
14 (Control) 0.34
B. Stirred-Jar Fermentors
The actual protein and cellulose content of the stirred jar fermentations
at a given moment were influenced by such factors as evaporation (concentrative
effect), which increased with the use of higher aeration rates, and the addition
of antifoamant (diluent effect) , if any.
Volumes removed for analyses were measured in order to determine
evaporation losses, but no correction was applied since these were a homogeneous
blend of the total volume present.
Thus, in order to relate the values back to the original volume,
correction factors were derived and applied to each case by the following method;
1. evaporation/day was computed,
2. the difference between the evaporation and the amount of antifoamant
added was found,
3. this difference (+ or -) was subtracted from (or added to) the
initial volume which gives the volume at that time.
4. volume at that time/initial volume=correction factor
5. (correction factor)(protein or cellulose content)=
corrected protein or cellulose content.
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Example: Fermentor #1 In experiment #12
1. Evaporation in 12 days * 610 ml
2. Antifoamant added in 12 days = 480 ml;
difference = -130 ml
3. Initial volume = 7530 ml;
7530 - 130 = 7400 ml
4. Correction factor = 7400/7530 = 0.983
5. Uncorrected Uncorrected Corrected Corrected
Protein Cellulose Protein Cellulose
Produced, Remaining, Correction Produced, Remaining,
Day ing/ml mg/ml Factor mg/ml mg/ml
12 1.44 7.88 0.983 1.42 7.75
MCJ652
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