close

Вход

Забыли?

вход по аккаунту

?

THERMOPHILIC FERMENTATION OF CELLULOSE

код для вставкиСкачать
ACKNOWLEDGEMENT
The author wishes to express his
sincere appreciation and gratitude to
Dr* R* W. Stone for his inspiring
interest and helpfulness during the
course of this research.
Page
6. The Effect of Calcium Carbonate
on the Rate of Fermentation and
Adjustment of pH to ,2 - 7 *^
during Fermentation
4-9
7 - Effect of Age of Culture on
Fermentation of Cellulose
52
S. Effect of Heat-Shock on the Rate
of Germination
C.
MORPHOLOGICAL STUDIES
53
5^
1. Liquid Medium: Morphological
Changes during Fermentation of
Cellulose
5^
2. Morphology on Solid Medium
5^
3 * Effect of Heat Treatment:
Morphological Changes during
Fermentationof Cellulose
D. BIOCHEMICAL REACTIONS
60
6l
1.
Media
6l
2.
Motility
62
3 . Flagella Stains
62
E. FERMENTATION OF OTHER CELLULOSIC
MATERIALS
66
F.
69
ANALYSIS OF FERMENTATIONPRODUCTS
1. Gas Analysis
69
2. Chemical Analysis
jG
a. Determination of titratable
acidity
76
b. Determination of reducing
sugars
77
c* Determination of volatile
solvents
77
d. Determination of volatile
acids
7&
Page
e. Determination of lactic
acid
7g
f. Determination of fixed
acids
72>
3 . Qualitative Chemical Analysis
a. Acetyl methyl carbinol
S3
b. Glucososazone
2>3
Gr. EFFECT OF TOLUENE AND SODIUM FLUORIDE ON
THE END-PRODUCTS OF CELLULOSE FERMENT­
ATION
&S
1* Effect of Toluene
S6
2.
93
Effect of Sodium Fluoride
H. EXPERIMENTS ON FIXATION OF ACETALDEHYDE BY THE SULFITE METHOD
101
I. ATTEMPTS TO TEST FOR THE PRESENCE OF
CELLULASE ENZYME
107
1 . Bacteria-free Filtrates
107
2• Wet Bacterial Cells
112
3 « Dried Bacterial Cells
115
Residue from Fermentation of
Cellulose
115
SUMMARY AND CONCLUSIONS
119
BIBLIOGRAPHY
12S
INTRODUCTION
AND
REVIEW OF LITERATURE
INTRODUCTION
Studies on the microbial decomposition of
cellulose and cellulosic materials in nature have
been subject to many investigations.
Because of the
vast amount of cellulose distributed in plant tissue,
and its subsequent culmination in plant waste, the
utilization of this substance through bacterial and
mold fermentation has attracted considerable
attention from both the scientific and practical
standpoint.
The decomposition of cellulose embodies
a series of chemical reactions catalyzed by enzymes
elaborated by microorganisms.
The participation
and the specificity of the microorganisms in these
chemical transformations depend upon the environ­
mental conditions and the chemical composition of
the substrate.
Because of the relative rapidity of
the decomposition of cellulose at higher temperatures,
the thermophilic bacteria are of more practical
importance under laboratory conditions than molds
and other microorganisms.
With these considerations in view, the object
of this investigation was concerned with (l) the
isolation and cultivation of thermophilic bacteria
2.
capable of breaking down cellulose,
(2) the factors
influencing the fermentation of cellulose,
nature of the products formed,
(3) the
(U-) studies on the
mechanism of cellulose breakdown, and
(5) attempts
to isolate the cellul&se enzyme.
REVIEW OF LITERATURE
As early as 1 & 99 > MacFayden and Blaxall
(c.f.
McBasth and Scales (l)) isolated bacteria from manure
which fermented cellulose at 60°0.
They attributed
the fermentation to the combined action of symbiotic
strains, and observed the presence of acetic and
butyric acids, but did not determine these quanti­
tatively.
Kellerman and his associates (2) introduced
the cellulose agar plate method for the isolation of
thermophilic organisms.
They noted that after iso­
lation of the bacterial colonies on agar plates and
inoculation into liquid cellulose medium, no fermen­
tation took place.
However, they observed that
previous inoculation of these colonies into sterile
sand rejuvenated their capacity to destroy cellulose.
McBeth and Scales (1) encountered the same
3difficulty and concluded that thermophilic bacteria
lose their cellulose-destroying power after one
transfer on a non-cellulosic medium.
Pringsheim
and Lichtenstein (3) have failed to get platepicked cultures to ferment cellulose.
Similarly,
Hutchinson and Clayton (if-) using the Kellerman agar
plate method were unable to ferment cellulose from
selected colonies.
Gray and Chalmers (5) obtained
negative results in similar studies.
By using the cellulose— agar plate method
Langwell and coworkers
(6, 7) have reported that
colonies from cellulose-agar incubated at room tem­
perature or at 37°C when inoculated into liquid cel­
lulose medium would produce fermentation at 65°C.
In these studies they have accounted for the complete
recovery of destroyed cellulose on the basis of endproducts found (hydrogen, methane,
carbon dioxide,
acetic acid, lactic acid, and ethyl alcohol), but
did not Teport a carbon balance.
These workers
claimed that by varying conditions the yields of endproducts could be changed.
Their results have not
been corroborated and the purity of the cultures used
in this investigation is questionable.
these experiments, Langwell,
Contrary to
in his patents, advocated
K
the use of crude cultures from manure rather than the
purified cultures used in the laboratory experiments.
It was reported by Kroulik
(2 ) that thermo­
philic cellulose fermenters are widely distributed
in nature.
In his investigations he described two
aerobic and two anaerobic species.
Because the aerobic
types failed to attack cellulose when isolated from
beef extract agar plates and since they were predom­
inant in the early stages of the fermentation, he
assumed that they were not cellulose fermenters.
The
ana.erobic organisms did not grow on beef extract agar.
As a result of the failure of separating these
organisms,
firmed.
the purity of the cultures was not con­
According to Kroulik some of the cultures
were able to withstand flowing steam treatment for
two hours.
butyric,
The end-products found were acetic,
formic and lactic acids,
carbon dioxide and
hydrogen.
From the intestinal flora of man Madame
Khouvine (9) isolated an organism which fermented
cellulose at 35 “
She believed this isolation
to be a pure culture and named it Bacillus celluloseae
disBolvens.
Ethyl alcohol, carbon dioxide, acetic
acid, and small amounts of hydrogen and butyric acid
5accounted for only about half of the carbon of the
cellulose*
The addition of sterilized fecal extract
or sterilized fermentation liquor aided the initiation
of the fermentation process.
Viljoen, Fred and Peterson (10) claimed to
have isolated a pure culture of a thermophilic organism
from manure which destroys cellulose at 65°C.
This
was made possible by a series of enrichment cultures
transferred at 5 day intervals*
The organism was
described as a gram negative, spore forming rod.
The
spores were able to withstand 115 °C for 35 minutes,
and heating them at 100°C for 5 - 1 0
the rate of germination.
minutes increased
Because of its morphological
character and ability to destroy cellulose rapidly at
high temperatures the organism was named Clostridium
thermocellum.
Colonies isolated from cellulose-agar plates
gave good growth at 65°C on agar slants, beef broth,
sterile soil containing filter paper, beef peptone
plus raw potato, raw banana, and yeast water.
But
in no case did the cultures regain their ability to
ferment cellulose when reinoculated into liquid cel­
lulose medium from any of the above media.
However,
by using a deep tube containing cellulose agar, these
6
investigators were able to isolate discrete colonies
that fermented cellulose.
In this manner they be­
lieved the isolation was a pure culture and identical
to that of the stock culture which had been subjected
to enrichment for almost two years.
The culture
showed no growth at 2&°C and 37°1-' In several media.
It was found that organic nitrogen in a form such as
peptone (best) was essential for the fermentation.
Acetic acid, butyric acid, ethyl alcohol, carbon
dioxide, hydrogen, and an ether soluble yellow
pigment were the end products determined.
The first report of the presence of a cellulosedestroying enzyme in a culture growing at 55°G was made
by Pringsheim (ll).
He believed that the enzyme loses
its activity soon after the death of the bacterial
cell and hence has never been found in a cell-free
extract.
By use of enrichment cultures and toluene,
growth of the organism was arrested and sufficient
glucose and cellobiose were accumulated to indicate
that these were intermediate products in the fermentation.
In another investigation Pringsheim (l2) presented
an analysis of the end-products which accounted for
^5 P er cent of the fermented cellulose.
The compounds
found were acetic acid, small amounts of formic acid, and
I
7large amounts of carbon dioxide and hydrogen*
Neuberg and Cohn (13) have demonstrated the
presence of acetaldehyde as an intermediate product
in the thermophilic fermentation of cellulose by
organisms isolated from canal mud and horse manure.
The workers confirmed the fermentation of glucose and
cellobiose by their cultures at 53 - 55°C since these
substances were shown to be intermediates of cellulose
i
fermentation.
Calcium sulfite, sodium sulfite, and
dimedon were employed as fixing agents for the quanti­
tative estimation of acetaldehyde;
sodium nitroprusside
and piperidine were used as colorimetric reagents for
the qualitative detection of acetaldehyde.
On studying the fermentation of cellulose and
fiber of certain feeds, Woodman (l^) and Woodman and
Stewart (15, 1 6) have concluded that the thermophilic
cellulose fermenters play an important role in the
utilization of cellulose by ruminants.
They were able
to confirm Pringsheim's experiments concerning the use
of toluene for the detection of glucose by the prepar­
ation of the osazone derivative.
Under normal con­
ditions cellulose was found to be almost completely
decomposed whereas with toluene added, only J>0 to
per cent of the cellulose was destroyed.
They believed
g.
that the microorganisms utilized the glucose formed
in a normal fermentation for metabolic purposes and
subsequently produce end-products such as organic
acids, alcohol, and gases;
whereas when toluene
was added at the “head*' stage of fermentation,
the
metabolic activities of the organisms ceased, and
glucose tended to accumulate.
Woodman and Stewart believe that the
mechanism of cellulose decomposition is resolved
into two stages, the first phase is dependent on
living microorganisms which are unable to utilize
the complex cellulose molecule directly for metabolic
purposes, but can elaborate enzymes which catalyze
the hydrolysis and initiate the process.
The second
phase involves enzymes which act on partially hydrolysed
cellulosic substances and convert them to glucose.
Further work by these investigators has shown that
whenever organisms were cultivated on cellulose-free
medium, they would tend to lose their cellulose
destroying capacity.
Coolhaas (17) k&s isolated thermophilic
cellulose fermenters from mud and manure.
Crude
cultures were found to produce methane and carbon
dioxide, but on subsequent enrichment by repeated
9
transfer only hydrogen and carbon dioxide were
formed.
By employing the cellulose agar plate
method, he was successful in isolating pure cultures.
However, when these were transferred to liquid
cellulose medium, no fermentation took place.
Because of this failure, he concluded that the problem
of cellulose fermentation remained unsolved.
By varying anaerobic conditions and applying
heat treatments, Tetrault (l$) obtained a change in
the flora of thermophilic cellulose fermenters.
Instead of the usual large rods of the plectridium
type he found very small rods predominating.
The products of thermophilic fermentation
of cellulose and the effect on the end-products of
purifying the cultures were studied by Scott, Fred,
and Peterson (19).
It was found that JO to SO per cent
of cellulose carbon was accounted for as acetic acid
(M-5 to 65 per cent), car bon dioxide, glucose, ethyl
alcohol, lactic acid, and succinic acid.
Some of the
undetermined carbon was considered to be converted
to a gum-like material soluble in water, but precipitable by acetone.
Glucose was demonstrated as an
end-product of cellulose decomposition by mass fermen­
tation.
The chemical analysis of its prepared osazone
proved the sugar to be glucose.
was not shown to be present.
Cellobiose, however,
It was assumed that at
55°C cellobiase is active ^hereas at the temperature
used (70°C) by Pringsheim only cellulase is active.
These workers showed that purification of cultures by
the enrichment method tends to increase the production
of acetic acid and glucose as an end-product, and
decrease the yields of alcohol and carbon dioxide.
Tomoda (20, 21) isolated thermophiles from
harse manure that fermented cellulose and cotton at
65°0.
Sawdust, plantleaves, and sulfite pulp were
not appreciably attacked.
The end-products and average
yields obtained from a two per cent filter paper medium
were;
16 per cent ethanol, J per cent butyric acid,
23 per cent acetic acid, one per cent lactic acid,
0.U- per cent hydrogen,
and 19 per cent carbon dioxide.
Methane was not found.
The first report on the thermophilic fermentation
of corn cobs was presented by Langwell (22).
A 6.2
per cent corn cob medium produced 7»75 P er cent
ethanol and ^0 per cent acetic acid.
Thick mashes
of corn cob medium were found to be difficult to fer­
ment by these microorganisms and cereal straws were
quite resistant to attack.
Langwell believed that the
11
presence of a high percentage of lignin in the straw
tended to retard the fermentation of cellulose*
Organic nitrogen such as peptone or yeast extract
and inorganic nitrogen such as ammonium sulfate were
found to be essential for the fermentation of corn
cobs*
Asparagine proved to be a poor form of organic
nitrogen.
W i t h adjustment of pH to 7 *° daily wi t h
sodium bicarbonate, Langwell obtained maximum
fermentation of cellulose*
Sodium bicarbonate was
preferred to calcium carbonate in the medium,
for
at lower p H values calcium carbonate forms calcium
acetate which ties up the phosphate ion as an
insoluble complex substance.
In using enrichment cultures of thermo­
philic cellulose fermenters, Snieszko (23) reported
yields of 50 P®r cent acetic acid and 13 pex\ cent
ethanol from 100 grams of cellulose fermented*
The
cultures were prepared by the enrichment and plate
methods and were considered to be pure.
Veldhuis, Christensen,
and Fulmer (2*0 used
crude thermophilic cultures from horse manure in order
to determine the possibility of ethanol production
from cellulose.
The organisms were assumed to be mixed
12.
cultures since on further enrichment they lost their
original fermentative ability.
They believed that
the breakdown of cellulose was due to a symbiotic
association of several bacterial species.
No
attempts were made to confirm the purity of these
cultures.
It was found that a temperature of 55°0
favored the highest yield of ethanol (26 per cent),
and a temperature of 60°0 favored the production of
acetic acid (24- per cent).
n - butanol and n - butyric
acid were also identified among the products of
fermentation.
By daily adjusting the pH at 7-75 to
S.O with sodium carbonate or dilute hydrochloric acid
the highest yields of alcohol were favored, and at
pH 7.50 to 7. 75 , acetic acid production was favored.
Cellulose concentration between two and five per cent
appeared to be best for the production of alcohol and
acetic acid*
These workers also studied the effect of
various concentrations of salts and report the follow­
ing optimum:
ammonium chloride 0.2 - 0 .4- per cent and
FeCl^ . 6H2O less than 0.1 per cent.
MgCl2 * ^ ^2^ ^ad
no effect in the concentrations used while traces of
tin were considered to be beneficial.
Aeration during
13
.
fermentation was found to decrease the yield of
alcohol and to a lesser extent, acetic acid.
A
substance giving a strong iodoform test was deter­
mined and believed to be acetone.
M an y contributions on the utilization of
lignin by microorganisms are reported in the literature.
Since lignin is intimately associated with cellulose
in many natural cellulosic products,
some of the recent
work has dealt with the effect of lignin on the
fermentation of cellulosic materials.
Acharya (25) in studies of mesophilic
fermentation of rice straw and other natural materials
found that the higher the lignin content of the
substrate the more resistant it was to decomposition.
In a publication reported by Peterson and
Snieszko (26) it was shown that thermophilic bacteria
did not attack various prepared w ood pulps to the same
extent.
Pure cellulose,
on the other hand, was readily
fermented by these organisms.
These investigators
thought that the nature of the union and the amount
of lignin tied up with the cellulose had a direct
influence on the susceptibility of cellulose to
decomposition.
Waksman and Hutchings (27) reported that the
14-.
destruction of lignin in oat straw and spruce wood
was slower than the other groups of plant constituents.
Free lignin decomposed faster than native lignin of
these plants.
Olson, Peterson and Sherrard (23 ) found
that enrichment cultures of thermophilic organisms
readily fermented pure cellulose,
but did not ferment
groundwood either alone or in the presence of filter
paper.
Holocellulose
free wood)
(total carbohydrate of extractive-
fermented readily.
'When added to a medium
containing free cellulose, lignin as groundwood (lignin—
containing material),
decomposed.
only the pure cellulose was
It was confirmed that good fermentation
(35 per cent) of wood materials is accomplished when
the lignin content is less than one per cent.
authors concludedfrom these results
The
that the relation
between lignin and carbohydrates is chemical and not
merely physical.
Virtanen (29) on the other hand,
found that
enrichment cultures of thermophilic cellulose-destroying
bacteria fermented 33*9 P©^ cent of the cellulose in
finely ground birchwood in 10 to 14- days at 6l°C.
finer the wood was ground,
of cellulose fermented.
The
the higher the percentage
The fermentation products
formed 7/ere chiefly acetic acid and ethyl alcohol with
15some formic and lactic acids.
When treatment of
spruce wood with sulfite was stopped at 65 per cent
of the initial lignin content, SO per cent of the
cellulose was fermented in S days.
These findings
tend to refute the conception that lignin and
cellulose are chemically united in the wood.
Simakova (30) reported a thermophilic
cellulose fennenter which produced propionic acid
as the main product with small amounts of valeric
and formic acids.
According to Soeters (31) vigorous fermen­
tation of cellulose is accomplished only with the
combined action of thermophilic bacteria and a
contaminating organism such as B. coli.
In a report published by Berl and Koerber (32),
it was shown that by varying the pH 6.0 to 7.^- at 55°C
the decomposition of cellulose increased from 20 per
cent to 70 Per cent.
Similarly,
the corresponding end-
products were produced under these conditions.
tives of carbohydrates,
Deriva­
like cellulose humic acid were
found to be resistant to the action of these micro­
organisms.
According to several investigators cellulose
appears to be attacked by an extracellular enzyme.
16 .
Nevertheless most attempts have failed to demonstrate
its presence in a bacteria-free filtrate.
Simola (33)
claimed he was able to show cellulose in a culture
fluid of Cellobacillus mvxogenes. After heating
bacteria and paper containing bacteria with toluene
and incubating at 37 °’-' ^o;c several days he obtained
an increase of a reducing substance as measured by
titration with permanganate solution.
In this manner,
Simola assumed that cellulase was present.
EXPERIMENTAL METHODS
AND
RESULTS
EXPERIMENTAL METHODS AND RESULTS
*
I
A - ISOLATION
The following medium of Viljoen, Fred and
*
Peterson (10 ) was employed for the isolation of
thermophilic organisms*
Peptone (Difco)................ 5*0 gm.
Na(NHij.)HPOij. . ^H20 ............. 2.0 gm.
KH2P 0i|_........................ 1.0 gm.
8
CaCQ^ ......................... 2.0 gm.
MgSOi). . 7H 20 .................. 0.3 gm.
CaCl2 ......................... 0.1 gm.
FeCl^ . 6H20
.......
trace
Distilled W a t e r .............. .1.0 liter
R e a c t i o n ...................... pH 7.2 to 7 »^
Large test tubes containing strips of filter
paper were filled to two-thirds volume with the base
medium and sterilized for 20 minutes at 15 pounds
pressure.
Each tube was inoculated with 1 gram of
rapidly fermenting horse manure, capped with aluminum
foil, and incubated at 60°G.
1- Enrichment methodAt the end of S days, fresh tubes of medium
were inoculated with 1 ml. of the crude cultures and
*
incubated at 60°C.
Repeated transfers of the cultures
to fresh medium were made every ^th day.
At the end
of the 2th enrichment culture, four transfers were
made every two or three days.
Fermentation of cellulose
was attempted at 37°» ^5°» and ^ 0 ° 0 >respectively.
In a typical fermentation gas bubbles began
to rise after 36 "to
hours.
Within 5 to 6 days gas
production rapidly increased and carried the paper to
the surface of the medium.
At this stage the paper was
decomposing to an amorphous and gelatinous state which
turned to a yellowish brown color.
Gras evolution ceased
after 6 to 3 days and the suspended substances gradually
settled to the bottom of the tubes together with some
unchanged cellulose.
In some tubes the residual cellulose
was impregnated with a bright yellow pigment and in others
there was just a faint discoloration with hydrogen sulfide
plus butyric acid odors.
As the enrichment procedure was
continued, these differential characteristics became
more pronounced.
After the 12th transfer, fermentation
began almost invariably from 13 to 24- hours following
inoculation and was completed in 4- to 5 days.
At this
stage three different types of fermentation were observed*
The first was characterized by the production of
hydrogen sulfide and butyric acid odors.
The second type
19
.
formed a bright yellow pigment without hydrogen
sulfide or butyric acid odors.
The third exhibited
none of these characteristics.
Each of these
fermentations produced gas during the initial stage
together with other volatile substances, and finally
formed an amorphous residue at the bottom of the
tubes.
The organisms were designated as cultures
1 , 2, and 3 respectively.
After two and one-half
years of cultivation with over ~$00 transfers in
liquid medium, the cultures maintained their usual
fermentation characteristics and did not lose their
capacity to destroy cellulose.
The cultures stored
in the original fermented liquor at ice box temperature
for IB months did not lose their power to decompose
cellulose upon subsequent transfers.
Repeated attempts to ferment cellulose at
37°C have failed for as long as 3 weeks of incubation.
At 4-5°C there was slight evidence of cellulose dis­
integration at the end of 2 weeks and at 50°C its
decomposition took place within S to 10 days.
2- Plate Method:
Since the enrichment method was used on the
cultures from horse manure, their purity was obviously
20.
questionable.
While sprae workers regard this tech­
nique to be sufficient for the isolation of pure
cultures many microbiologists believe that mixed
cultures may result.
Therefore, the plate method
was used in attempts to isolate colonies of each
culture on solid media and to ascertain the ability
of the isolated culture to ferment cellulose.
The following culture media were used for
plating
agar,
(a) standard nutrient agar,
(b) glucose
(c) glucose yea,st extract agar, and
lulose agar.
(d) cel­
The reaction of each was adjusted to
pH 6.£>, J » 0 , and 7 *2 ,
(a)
Standard Nutrient Agar
Peptone
(Difco) ......
Beef-extract (Difco)
A g a r ..........
Distilled water
(b)
•
5.0 gm.
3.0 gm.
13*0 gm.
1.0 liter
Grlucose Agar
Nutrient agar
0.5 per cent glucose
(c)
Grlucose yeast extract Agar
Yea.st extract * ............... .100 ml.
Grlucose.........................5.0 gm.
Peptone (Difco)................5.0 gm*
A g a r .......................... 15.0 gm.
Distilled w a t e r ..............
(d)
900 ml.
Cellulose Agar
Liquid cellulose base medium
4* 1.5 '
Agar 4- 0.5 7° ground cellulose
*
Place one-half pound of moist pressed yeast in a
3 or 4 liter flask.
Add 2 liters of water and steam
for J to k- hours with occasional stirring.
The
infusion is then autoclaved for at least 4-5 minutes
at 15 pounds pressure and allowed to stand for 1 to
2 weeks.
The supernatant liquid is carefully
siphoned off and used without filtering.
ijt
Five grams of filter paper were triturated with
sand and water in a mortar.
The mixture and J 00 ml.
water were added to a large beaker.
The suspended
paper was poured off to the base medium and J 00 ml.
of water were added again to the sand-paper mixture
and poured off to the medium.
The volume of water
was then brought to 1 liter.
Suitable dilutions of active cultures were
prepared in sterile water blanks and plated out
aerobically and anaerobically with the above media.
Anaerobic conditions were furnished by the use of
alkaline pyrogallol solution in Spray dishes.
Half
of the plates were incubated at 37°c and- "t*1® other at
60°G for 2 - 3
days.
Observations were made at 2^,
4-S, and J 2 hour intervals.
At the end of the incubation period,
tubes
of liquid medium were inoculated from colonies of
these plates.
To some tubes sterile one per cent
glucose and one per cent cellobiose solutions were
added separately.
Slants of similar media were
inoculated from colonies of 60°C aerobic plates and
incubated at 60°G anaerobically.
Similarly inoculations
were made from anaerobic plates to aerobic slamts.
same procedure was used with the 37°^ plates.
In another series the 37 °^ cultures were
incubated at 60°C and vice versa.
In all cases the
incubation period was from 2 to 3 days.
Tubes of liquid cellulose medium were then
inoculated from each series and incubated at 60 °C.
One per cent glucose and one per cent cellobiose
solutions were added to some of these separately.
The
23Representative cultures from each series were kept
in an ice box for a month and tested frequently
for their cellulose decomposing power.
Glucose
and cellobiose solutions were used on these cultures.
Likewise, 10 month old cultures on plain agar and
dextrose agar slants were tested.
After IS to 2 ^- hours at 60°0 moist trans­
parent, pin-point colonies developed in both the
aerobic and anaerobic plates.
There was no noticeable
difference of growth observed in the media at pH 6.S,
7 .0 , and 7 -2 .
Standard nutrient agar, glucose agar,
and cellulose agar produced better growth than glucose
yeast extract agar.
After 2 days of incubation,
colonies when inoculated in the liquid medium produced
active fermentation of cellulose within 4 to 5 days.
Addition of glucose or cellobiose solution to the
fermentation tubes appeared to stimulate their growth.
Growth was scanty on the 37°c plates after 2 days of
incubation.
However after 3 days,
the colonies were
similar to those of the 60°^ plates.
Fermentation of
cellulose took place within ^ to 5 days when picked
colonies were used for inoculums.
With glucose or
cellobiose solution added the stimulatory effect in
the fermentation of cellulose was again noticeable.
Inoculation of aerobic slants from anaerobic
plates and from aerobic plates to anaerobic slants
produced similar growth, in 2 to 3 days at 60°C.
In
both cases growth was characterized by a filiform,
somewhat beaded, and transparent appearance.
Cellulose
was fermented by these cultures in 4 to 5 days.
With
an analogous procedure at 37°^> similar results were
obtained.
After 3 days of incubation cellulose was
attacked within 4- to 5 days by each culture.
Corres­
ponding results were also obtained when cultures at
60°C were incubated at 37°^ &nd vice versa.
Again
glucose and cellobiose were found to influence the
rate of cellulose fermentation when added to the
liquid medium.
This stimulatory effect was more
pronounced when older cultures were used and with
certain of the media..
One month old cultures from
nutrient agar and glucose agar showed more rapid
decomposition of cellulose by 2 to 3 days than those
from cellulose agar medium.
Ten month old cultures from plain and cellulose
agar produced only slight growth in the liquid medium
with no decomposition of cellulose after 10 days, but
with added glucose or celldhiose the cellulose was
attacked in ^ to 6 days.
On the other hand, cultures
25.
from glucose agar slightly decomposed cellulose
in &
to 10 days and with added glucose or cellobiose,
fermentation ended in 4- to 5 days.
the
In most cases,
glucose was found to be more stimulating than cello­
biose.
3” Deeo-tube semi-solid agar method:
Large test tubes were filled to two-thirds
volume with the cellulose medium described under plate
methods.
In this medium one per cent agar was used
instead of 1*5 Pe£ cent.
In another series the same
medium was used except that three long strips of
filter paper were added to each tube instead of ground
filter paper.
Sterilization ws.s at 15 pounds pressure
for 20 minutes.
Suitable dilutions of active cultures were
prepared with sterile water blanks and one ml.
inocu­
lations were made into the medium at ^4-5°^ s-nd rapidly
cooled.
The tubes were capped with aluminum foil and
incubated at 60°C for a week.
At the end of this
period attempts were made to isolate colonies and to
transfer them into the liquid medium.
Due to gas production the agar in the deeper
portions of the tubes was split after two to three days
of incubation.
Culture 1 which produces hydrogen sulfide
and butyric acid formed a black sediment, culture 2
developed the usual yellow pigment,
and culture 3
did not show any of these properties.
The colonies
in each case were very small and transparent.
Those
colonies growing in tubes containing paper strips
were too concentrated around the paper to single
out for transplantation.
However,
from the ground
paper medium the colonies were not as crowded and
thus were more easily transferred to the liquid
medium.
Fermentation of cellulose took place in
5 days•
4— Successive Transfers:
Each culture was transferred daily into
fresh liquid medium for 25 days and incubated at
60°0.
Observation of each transfer was made for
extent and time of completion of fermentation.
In every instance fermentation was ended
in ty- to 5 days.
Cellulose was decomposed without
any significant difference.
Variations were noted
in pigment formation, hydrogen sulfide and butyric
acid odors.
The typical yellow pigment of culture 2
was less pronounced in the last 25 transfers.
Similar­
ly, the odors of hydrogen sulfide and butyric acid
27.
n
of culture 1 were still present after the l4-th sub­
culture.
However, when transfers were made of each
culture after 5 days of fermentation all the character­
istics of the original, culture returned.
did not change in any of the transfers.
Culture 3
The fact
that fcach culture fermented cellulose after 25
successive one-day transfers was indicative of its
ability to attack this substrate.
Although a
diminution of their cha.racteristics was obtained,
subsequent transfers fully restored their original
qualities.
5~
Purification of Cultures by Heat Treatment:
Tubes of sterile liquid cellulose medium were
placed in a 100 - 105°C oven.
within the tubes reached 100°C,
was introduced into each.
5 days old were used.
6 hours,
When the temperature
one ml. of culture
Cultures 1 , 2 , 3>
and
At half hour intervals for
sets of tubes were removed from the oven,
cooled and incubated at 60°C.
Similarly the 2 week
old cultures were heated, for 6 hours at 100 - 105°C.
One ml. transfers were made each half hour into
fresh medium.
After heating for 30 minutes,
decomposed cellulose in ^ to 5 days.
all cultures
When heated for
6 hours,
the fermentation of cellulose was ended
in 8 - 10 days by 1, 2, and 3 day cultures and in
6 to 7 days by U- and 5 day cultures.
The time to
complete fermentation was correspondingly lower
by each culture when heated less then 6 hours.
The
odor of hydrogen sulfide plus butyric acid of
culture 1 and the yellow pigment of culture 2 were
wealcly discernable in the 1, 2, and 3 day cultures.
However,
on subsequent transfers these characteristics
were appreciably intensified.
In the case of culture 3,
there was no significant difference noted between the
control and heated cultures.
Fermentation of cellulose
was completed in 5 to 6 days by 2 week cultures when
heated for 6 hours.
In each case when transfers
were made to new medium the fermentation was ended
in k- to 5 days.
Although it is impossible to say positively
that the cultures were pure,
the characteristics
observed in the tests just described lend strong
support to that contention.
Heating the cultures
for 6 hours at 105°C would obviously kill any non­
spore forming organism present.
Thus the cultures
are limited only to spore forming rods.
The following
observations are evidence that each culture is probably
p ure.
29.
First, repeated isolation of single colonies
from agar gave the same characteristic fermentation.
Second, various heat treatments on cultures
of different age levels did not significantly alter
the fermentations when compared with the mother
cultures,
if one allows for the fact that the heat
treatment was much more severe on the young cultures.
Third,
incubation of the cultures at various
temperatures although causing a difference in rate
of fermentation never changed the morphology of the
organisms nor the essential type of dissimilation.
If more than one organism was present,
it is logical
to expect that changing growth temperatures would
favor one more than the other.
Fourth, cultivation of the organisms under
radically different conditions of anaerobiosis did not
materially effect morphology or type of fermentation.
Fifth, long holding in the ice box had no
marked effect.
Sixth, the ability of each culture to ferment
cellulose after 25 successive one-day transfers without
loss of original characteristics may be attributed to
the mechanism of a pure culture.
30
Seventh,
in all the above conditions and
various combinations of them, the original fermen­
tation could always be obtained by placing the
cultures under standard conditions of medium,
temperature, and anaerobiosis.
Therefore,
it is
assumed that the three cultures as described are
each composed of only one organism.
t
.
31*
B- CULTIVATION
1- Medium:
Five different media were employed in a
series of experiments to study their effect on the
extent and rate of cellulose decomposition.
Each
medium differed mainly by the source of nitrogen.
The following media were employed:
A— Omeliansky's medium:
Ammonium sulfate
as inorganic source of nitrogen
K 2H P 0H ........
1.0 g m .
(NHi|.)2S0ij......
1.0 gm.
MgSO^ . 7H 20 ..
0.5 gm.
N a C l ..........
trace
C a C o - j .........
2*0 g m .
Distilled water
1.0 liter
A-l-
Omeliansky's medium plus 5 grams of
Difco Peptone.
A— 2- Omeliansky's medium plus 100 ml.
of
yeast extract and distilled water to make 1 liter.
A - 3- Omeliansky's medium plus 100 ml. horse
manure extract and distilled water to make 1 liter.
Preparation of horse manure extract:
Fifty grams of horse manure and ^>00 ml. distilled water
32
were mixed in an Erlenmeyer flask and heated in an
Arnold for half an hour.
The mixture was filtered
through a Buchner funnel several times until no
sediment was present in the filtrate and distilled
water was added to make one liter.
The extract was
sterilized for 30 minutes at 15 pounds pressure.
B - Viljoen,
Fred, and Peterson medium (10):
Microcosmic9 salt as source of inorganic nitrogen.
. 4-H20 ............. 2.0
gm.
K H 2P Q ^ .......................... 1.0
gm.
CaCO-^ ........................... 2.0
gm.
MgSO^ . 7H20 ......... ......... 0.3
gm.
CaCl2 ........................... 0.1
gm.
NaUH^HPO^
FeCl-j . 6H20 ................... trace
Distilled water ............... 1.0 liter
Media B— 1 , 2 , and 3 have the same additions
■A
to the base as listed under A-l,
2, and 3 respectively.
C- Nitrogen-free basic medium.
M g C l 2. 6H20 . .................... O .3 gm.
K 2H P 0^ .
M g C 03
..1.0 gm.
0.3 gm.
CaCO^... ..........................2.0 gm.
33NaCl ............................ trace
FeCl-j . 6H20 ............ .
trace
Distilled water ............... 1.0 liter
Media 0-1, 2, and 3 ba-ve the same additionc
to the
base as listed under A-l, 2, and 3 .
D-Ammonium nitrate and
microcosmic salt
medium.
CaCO^ . ........................... 2.0 gm.
KgHPOij............................. 1»0 gra.
MgCO^
0.1 gm.
Ha(NHif)HP0l{.
NaOl
.4-H20 .............. 0.5 gm.
..........................trace
1.0 gm.
Distilled w a t e r
*...1.0 liter
Media D— 1 , 2, and 3 have the same
to the
base as listed under A-l, 2, and 3>
additions
respectively.
E- Ammonium chloride and microcosmic salt
medium.
N H ^C l ............................. 1.0 gm.
CaOO-^
2.0 gm.
ligCO-j
0.1 gm.
K 2H P 0 ............................ O .5 gm.
Na(NHij.)HPO}^
.*iH2Q .............. 1.0 gm.
3^.
CctClg
0.5 gra.
.>
FeCl-j . 6H20 . .
trace
Distilled water
1.0 liter
M e d i a E-l,
2, and 3 have the same additions
to the base as listed under A-l,
2, and 3 respectively.
E ach medium was adjusted to pH 7.2 - 7 .^
and prepared in large tubes as previously described.
One ml. of each culture was used as inoculum and
incubation was at 60°C.
daily for two weeks.
Observations were made
Results are given in table I.
35Table I
Days Necessary to Complete Fermentation
* - incomplete decomposition
** negative decomposition
Culture 1
Davs
Medium
Culture 2
Davs
>#*
A
A-l
1-6
A-2
Culture 3
Davs
Ik-**
6 - .7
6 - 7
6 - 7
6 - 7
6 - 9
A-l
14-*
li^t.
14-*
B
1^**
14-**
B-l
..
B— 2
__
...
4- - 5 ...
. 5 - 6
B - l ____
14-*
C
14-*
14-*
14-**
14-**
9 -11
C-2
14-*
c-i
14-*
Ik-*
14-*
D
Ik-**
l4-**
14-**
D-l
6 - 7
7 - 6
7 - 6
D— 2
7 - 6
7 ...-. 6
9-10
D-l
14-*
1*4-*
14-*
E
Ik-**
E —1
5 - 6
E-2
7 - 6
7 - 6
9 - 1 0
E-l
1I4*
14-*
14-*
i
9-11
I
!
6 - 7
1
5 - 6
H
0
1
H
ro
i
i
i
6 - 10
k- - 5
OJ
H
1
O
H
C-l
k- - 5
14-**
...
6 - 7
.... 6 - 7
36.
Table I indicates that in every case
the medium devoid of organic nitrogen did not support
growth.
Manure extract supported growth with partial
decomposition of cellulose, but usually the fermentations
retained only a few characteristics of the original.
Yeast extract medium sustained growth in most cases
but in some the fermentations appeared to be sluggish.
Peptone was found to be most stimulating and usually
gave vigorous fermentation.
Medium C with no inorganic
nitrogen or sulfur produced the most sluggish fermen­
tations.
Media D and E with inorganic nitrogen but
no sulfur gave better results.
were produced by medium E.
More active fermentations
Hydrogen sulfide and
pigment formation were not as evident with these
media as with A and B which contained both inorganic
nitrogen and sulfur.
Although media A and B gave
almost parallel fermentations, the latter proved
superior in giving consistent results.
From these results it would appear
that the presence of inorganic nitrogen and sulfur
are necessary for best fermentation and that ammonium
nitrogen is more effective than nitrate nitrogen.
Media devoid of these elements developed only slight
pigment and faint odors of butyric acid and hydrogen
sulfide.
Such fermentations were usueJ-ly sluggish and
further cultivation on similar m e d ia reduced the
activity still further.
2- Effect of Organic Nitrogen Source.
The following experiments were conducted
to determine the relative effects of several organic
substances upon cellulose decomposition.
Sterile solutions of Witte, Merck, and Difco
Peptone, asparagine,
and cysteine were prepared.
The
rpedia used were those listed under Cultivation and
designated as A, B, and E.
The following solutions
were added to tubes of media in the concentrations
indicated below.
I
0 .5% peptone 4- 0.01$ cysteine
0 .5% peptone 4- 0.02$ cysteine
0.5$> peptone 4- 0.0$$ cysteine.
II
0 .5# peptone + 0.01$ cysteine 4. 0 . 0 5$ asparagine
0 .5%’ peptone 4- 0.02$ cysteine 4- 0.05/» asparagine
0.5$ peptone 4- 0.0$$ cysteine
III
0 .05$ asparagine
0 .01$ cysteine 4- 0.05$ asparagine
0 .02$ cysteine 4- 0 .05$ asparagine
0 .0k-fo cysteine 4- 0 .05$ asparagine
IV
0 .5$ peptone 4- 0 .05$ asparagine
33
V
To serve as controls 0 .5$> peptone,
0 .0 2 , and 0 .0^
cysteine,
*
0.01,
and 0 .05$ asparagine were
employed separately in the media.
VI
Media devoid of organic nitrogen were
used.
The tubes were inoculated with 1 ml. of
culture and incubated at 60OC.
Observations were
made daily for 2 weeks.
When cysteine was used alone or with
asparagine, the initial growth was observed in 7
to 10 days.
However,
the fermentations appeared
*
sluggish and only slight decomposition of cellulose
occurred at the end of two weeks.
The rate of
growth was not markedly influenced by the concentra­
tion of cysteine.
Hydrogen sulfide and pigment for­
ma,tion were hardly discernible.
asparagine alone support growth.
In no case did
When peptone
either alone or with any of the designated combinations
was used,
fermentation was always complete.
No growth
was obtained from any medium free of organic nitrogen.
Medium E free of inorganic sulfur was not able to
prbduce as much hydrogen sulfide with cysteine as with
peptone.
A surprisingly significant difference in
fermentation was observed with Difco, Merck,
and Witte's
a
peptones.
The first promoted vigorous fermentation
in 3 to 5 days and the latter two supported only
slight growth without decomposition of cellulose
after two weeks of incubation.
When subsequent
transfers of the cultures were made to similar
media, no growth was obtained at the end of two
weeks, but when transferred to a medium containing
Difco peptone,
the original fermentation was restored.
Whether this inhibitory influence on the rate of
fermentation may be attributed to toxic substances
in the peptone or certain stimulatory compounds in
the Difco peptone has not been determined.
This
behavior ma.y also be a reflection of the nitrogen
requirements which are essential for metabolism.
The marked differences
in fermentation noted between
inorganic nitrogen and various organic nitrogen
substances have further differentiated the nitrogen
requirements of the organisms.
the form of peptone
Organic nitrogen in
(Difco) was found to be best for
growth and promoting vigorous fermentation.
3• The effect of salts at low concentrations:
The following sterile sa.lt solutions were
employed to determine their effects on cellulose break­
down:
ferric chloride,
aluminum chloride,
zinc chloride
40.
ferrous sulfate,
stannous chloride,
barium chloride,
lanthanum chloride,
perborate.
lithium chloride,
and sodium
In addition, boric acid was used.
Media A-l and A-2 (minus sodium chloride) and B— 1
and B-2 (minus ferric chloride) were used.
Bach salt solution was added to tubes of
medium to concentrations of 0.01 and 0.02 per cent
respectively.
Controls of each medium were run.
One ml. inoculum was used and incubation was at 60°C
for 2 w e e k s .
Stannous chloride and ferric chloride were
found to be most stimulating in producing vigorous
fermentations.
There was no appreciable difference
noted between 0.01 and 0.02 per cent concentrations
for all the fermentations ended in 4 to 6 days.
Media devoid of these salts promoted growth but the
time of fermentation was extended 2 to 3 days.
chloride, manganous sulfate,
ferrous sulfate,
Zinc
and
sodium chloride influenced the rate of fermentation
to a lesser degree regardless of concentration.
The
fermentation period was similar to that of the
controls which were free of added salts.
chloride,
perborate,
Lanthanum
lithium chloride, barium chloride,
sodium
and aluminum chloride exerted a slight
ij-1.
retarding effect on the fermentations.
chloride,
With aluminum
sodium perborate, and boric acid,
the
fermentations were not completed in 2 weeks and
resembled the original cultures only slightly.
Peptone and medium B were found again to be superior
to yeast extract and medium A, respectively.
subsequent experiments medium B-l
In all
(same as under
isolation) was used where a liquid medium was employed.
Effect of atmospheric o x y g e n :
In the following experiments,
tests w ere made
to determine the effect of the presence or absence of
atmospheric oxj'gen on the relative decomposition of
cellulose using medium B-l.
Large tubes containing about 14-0— 50 ml.
and
agglutination tubes containing 3 rol. of medium were
inoculated with one ml.
respectively.
and one loopful of culture
A set of small tubes was placed in
Thunberg tubes and evacuated.
Another set of small
tubes and a set of large tubes were evacuated in a
dessicator by a vacuum pump and the remaining oxygen
was absorbed with alkaline pyrogallol.
A set of both
small and large tubes served as controls under atmos­
pheric conditions.
Incubation was at 60°C for one week.
The experimental procedure mentioned under plate methods
was used in this study.
M-2
There was no relative difference observed in
extent or type of fermentation regardless of oxygen
conditions.
The presence or absence of atmospheric
oxygen did not alter any original characteristics of
the cultures.
H- to 5 days.
All fermentations were complete in
It was observed that aerobic plates
and anaerobic plates at 37°^ and 60°0 produced
similar growth which on subsequent transfers to
liquid medium developed the usual fermentation of
cellulose.
Further transfers from colonies on plates
to slants at similar and reversed oxygen conditions
gave identical growth.
These cultures fermented
cellulose without loss of any characteristic property.
Because of the ability of each culture to
grow equally well and ferment cellulose under aerobic
and anaerobic conditions,
the organisms are considered
to be facultative anaerobes.
However, no chemical
data are available to determine which condition
favored more destruction of cellulose.
Furthermore,
it is recognized that even in the small tubes exposed
to air, once growth has been initiated,
the conditions
below the surface of the liquid will be relatively
anaerobic.
^3
5~ Hydrogen ion Concentration Studies:
a- Progressive changes of pH during
fermentation of cellulose.
In previous experimental procedures the initial
pH of the liquid cellulose medium was adjusted to 7*2 7.*4-.
The hydrogen ion changes during the fermentation
of cellulose were of interest in order to determine
the stage at which cellulose was decomposed.
In the following experiments pH measurements
were made by the potentiometric method (glass electrode)
every 12 hours during fermentation.
The pH of the
sterile medium was determined before and after
inoculation.
Large tubes were inoculated with 1 ml.
of culture and incubated at 60°C for 5 days.
44
.
Table II
pH Changes during Fermentation of Cellulose
p H Changes
Time in
Fermentation
Culture
Culture
Culture
hours________ Changes___________ 1_________ 2_________ 3
.7.4-3..
7.4-3 ..
7_. 4-3
7.36.
7.31
7. 26
6.96 .
7.01
6 .S3
6.73
6.67
6.4-7
Gas evolution
Slight decomposition
Paper carried to
top of medium
more decomposition
6.19
6.11
5.90
5*9^
5.91
5.73
72
Vigorous
fermentation
5.67
5.60
5.4-6
S4-
Decrease of
eas evolution
5.76
5.75
5.36
5.64-
5.72
5-35
VJl
•
pi
_pr
0
5.70
5.33
3.32
5.70
5^ 3.3_..
12
2436
4-g
60
96
106
120
Turbid
G-as evolution
more gas
evolution
Less gas evolution
sediment at bottom
Ho gas evolution
Fermentation
Complete
ii
4-5.
The progressive changes of pH during fermentation
of cellulose are given in table II.
Since the initial
pH at the first evidence of cellulose decomposition
varied w i t h each culture,
the corresponding optimum
pH was accordingly different.
It is noted that each
culture required J2 hours before active fermentation
took place.
The optimum pH range at which cellulose
was most vigorously attacked by culture 1 and 2 was
between 5*^0 and 5*50,
by culture 3 between pH 5.4-6
to 5*33.
Various optimum pH values have been recorded
in the literature.
Woodman and Stewart (l 6 ) and
Pringsheim and Liebowitz
(34-) reported that the
optimum pH value of the thermophilic decomposition
of cellulose was 4-.S and 5*40 respectively.
In study­
ing the breakdown of cellulose at 37°C, Bradley and
Rettger
(35) found the optimum pH to be at 4-. 6 to 4-.S
under aerobic conditions and Cowles and Rettger
(36 )
found it to be at pH 5.6 to 6.4 in anaerobic fermenta­
tions.
Grrassmann and Rubenhauer
(37) ancI Karreth (3 B)
recorded in patents that the optimum pH value of
cellulose activity was at 5*20 and 5*30 respectively.
Trager (35) isolated a cellulase enzyme from the
intestines of termites and roaches which decomposed
cellulose best at pH 5 .3 0 .
Because of these variable pH values the
relative differences may be attributed to the environ­
ment and the type of organism employed.
Consequently,
it may be considered that probably there is no fixed
pH value of optimum cellulose enzyme activity but
that it falls within the range of 5*0 to
f°r
most organisms.
•to- The influence of initial hydrogen ion
concentration upon the rate of cellulose decomposition.
Medium B-l was prepared in deep tubes and
adjusted to the following pH levels;
7 .*12, 7-2*4-, 7-05,
6 .7 5 , 6 .*4-0, 5.92,
7
and 5-50.
Colorimetric and potentiometric methods were used
to check the pH.
The tubes were inoculated with
1 ml. of culture and incubated at 6 0 °C.
were made dail3>- for 10 days.
Observations
*7 Table III
Effect of pH
of Medium on
Fermentation of Cellulose
Days Necessary
to complete
fermentation
7.^0
7.56
■5-6
__ _4 - 5
2
Days necessary
to complete
fermentation
4- 5
Culture 3
!
pH
Culture
1
CT\
Culture 1
ji
Medium
Days necessary
to complete
fermentation
7- &
.4.- .5
7.42_____ 2k.-_5._________ t_n_5_________ 4_- 5
6.75
6.40
5.92
5.50
Ln
l
cr\ <
T-.24_____ 4 -.5_________4 - 5 _________
4-5
7. OR
4- 5
4- 5
6 - 7
Incomplete
after 10 days
Incomplete
after 10 days
Incomplete
after 10 days
slight
decomnosition
No
dec ornpo s i t ion
after 10 days
Incomplete
after 10 days
slight
decomposition
No
decomposition
after 10 days
5 - 6
& -10
Incomplete
after 10 days
slight
de cornno s i t ion
No
decomposition
after 10 days
Ij-g.
The rate and extent of cellulose decomposition
was influenced by the initial pH of the medium as
indicated in Table III.
Each culture grew vigorously
and attacked cellulose when the initial pH was between
6.75 a-ttd
in
this range fermentation was ended
to 5 days, and at pH
6.^+0 and 5*92 fermentation
was incomplete and appeared sluggish after 10 days.
Although growth was supported at pH 5«50> no decomposition
of cellulose was observed.
Despite the fact that the
initial pH of the medium was within the realm of the
optimum pH range, fermentation of cellulose was either
incomplete or distinctly nega,tive.
The results emphasize
the apparent deterrent action of a slightly acid reaction
of the medium.
The inability of the cultures to ferment
cellulose effectively below pH 6.75 may
to several factors.
attributed
Since the buffering effect of
calcium carbonate is centered around pH 6 .5 , the
adjustment of the pH to lower values probably reduced
its capacity to neutralize acid.
Therefore,
the
actual pH during fermentation may have dropped quite
rapidly and the retardation of their mechanism may be
due to the increased acid.
Since best growth was
obtained when the initial pH was above 6.75 and cellulose
breakdown was found to be most pronounced below this
^9.
value, the fermentation may take place in two stages:
first, an initial growth of organisms and the elaboration
of a compound or compounds essential to cellulose
hydrolysis and second,
the actual breakdown of cellulose
at a lower pH.
6-
The Sffect of Calcium Oarbonate on the
Rate of Fermentation and Adjustment of pH to 7.2 - 7.4during Fermentation:
'Two separate media were prepared in deep tubes
with and without calcium carbonate at pH 7.2 - 7-4-.
Brom thymol blue indicator was added to half of each
medium.
One ml. of culture was used as inoculum and
incubation was at 60°C for £ days.
Observations
were made daily for pH changes and cellulose decom­
position.
Adjustments of pH to 7*2 “ 7»4 were made
with sterile sodium hydroxide solution as acidity
appeared.
Controls of each medium without pH adjust­
ments were run simultaneously.
50.
Table IV
The Effect of Calcium Carbonate and
Adjustment of pH on the Rate of Fermentation
Medium
Control
CaCO-^
Control
No CaCO-j
CaCO-^ 4Brom thymol
blue
No CaCO-z
Brora thymol
blue
Culture 1
Bays
necessary to
complete
fermentation
k - 5
5 - 6
sluggish
- 5
5 - 6
sluggish
Culture 2
Days
necessary to
complete
fermentation
ij. _ 5
5 - 7
sluggish
U- - 5
6 - si
sluggish
CaCO-j 4Slight
Slight
Brom thymol decomposition decomposition
blue
Incomplete
Incomplete
pH adjusted after g days
after S d ays
Slight
Slight
CaCO -7
decomposition decomposition
pH adjusted
Incomplete
Incomplete
______ „ _____ after 8 d a y s af ter 8 days
No CaCO -7
Growth Growth Brom thymol
but no
but no
blue
decomposition decomposition
pH adjusted after g days after~ S days
Growth —
Growth —
Ho CaCO-^
but no
but no
pH adjusted decomposition decomposition
_____________ after 8 days
after S days
Culture 1
Days
necessary to
complete
fermentation
U- -
5
6 - g
sluggish
4. - 5
5 - g
sluggi sh
Slight
decomposition
Incomplete
after 8 days
Slight
decomposition
Incomplete
after S days
Very slight
decomposition
Incomplete
after 8 days
Very slight
decomposition
Incomplete
after S days
51
.
The normal fermentations with calcium carbonate
and without adjustment of pH show the usual rate of
decomposition of cellulose (Table I V ).
Although the
medium devoid of calcium carbonate supported fermentation
and required 2 to 3 days longer to complete the process,
there was a marked difference observed in the character­
istics of each culture.
Such properties as evolution
of gas, pigment formation, and odors of butyric acid
and hydrogen sulfide were scarcely discernible.
The
amount of cellulose fermented when compared to that
of medium with calcium carbonate was significantly smaller.
When the media were adjusted to pH 7.2 - "7.Uduring fermentation,
there was only slight decomposition,
of cellulose in the medium containing calcium carbonate
and practically no decomposition noticed in the medium
without carbonate.
In each case where pH was adjusted,
the cultures resembled the controls only slightly.
Brom thymol blue did not exert any deterrent action
on the rates of fermentation.
The inability of the cultures to continue
fermentation and decompose cellulose after adjustment
of pH to 7 *2
further evidence that the optimum pH
for breakdown of cellulose is much lower than 7»
Such
behavior is indicative of a mechanism which may be
dependent on two separate stages.
It appears that the
52.
first stage of the fermentation process is initiated
best in a neutral reaction and the subsequent stage
preconditioned by the former will only proceed vigor­
ously within the realm of the optimum pH range (5*0 ~
5 .$).
The fact that sluggish fermentations were
obtained in the absence of calcium carbonate emphasizes
the influence of adequate buffering power in producing
a vigorous reaction.
7-
Effect of Age of Culture on Fermentatio
of Cellulose:
Cultures 1 to 7 days and 1 , 6 , 1 2 , and IS
months old were used in these tests.
Each culture
was inoculated from its original fermentation liquor.
The old cultures were kept in an ice box.
Deep tubes
were inoculated with 1 ml. of culture and incubated
at 60°C until the end of the fermentation.
The rates of cellulose decomposition were
parallel in all ca.ses except those of the 12 and l£
month cultures.
Fermentations were completed in 4-
to 5 days in the former case and in 5 to 7 days in
the latter which in subsequent transfers decomposed
cellulose completely in 4 to 5 days.
A deeper yellow
pigment and stronger butyric acid plus hydrogen sulfide
odors characterized the older cultures.
Young cultures
(l and 3 days old) resembled the older cultures only to
53.
a slight degree and *4- day - 6 month old cultures to
a moderate extent.
S—
Effect of Heat-Shoclc on the Rate of
Germination:
Cultures varying in age from one day to
six months were placed in an oven at 100 - 105°C
for 15 minutes.
Fresh tubes of medium were inoculat­
ed and incubated at 60°C.
Observations were made
daily until fermentation was complete.
The stimulating effects of heat-shock on
the rates of fermentation varied between young and
old cultures.
Heat treatment did not affect the
rates of cellulose decomposition of the 1 to 6
day old cultures.
Older cultures after heat-shock
fermented cellulose in 3
4- days and in some
cases fermentations were completed in less than
3 days.
Without heat treatment old cultures at­
tacked cellulose in 6 to S days.
The difference
noted between young and old cultures was in the
intensity of the pigment and odors of hydrogen
sulfide and butyric acid.
In general,
older cul­
tures produced more vigorous fermentations with more
deeply colored pigment and stronger odors than
younger cultures after heat-shock.
The fermentative
5^.
characteristics of the old cultures closely resembled
those of the original enrichment cultures.
C - MORPHOLOGICAL STUDIES:
1“ Liquid Medium: Morphological Changes
during Fermentation of Cellulose:
Deep tubes of fresh medium (B-l) were inoculated
with 5 day cultures and incubated at 60°C for 5 days.
Smears were prepared daily before and after fermentation
and stained by the Gram, Kopeloff, and Fulton-Schaeffer
methods.
Twelve day and IS month cultures were used
and stained similarly.
Three sets of agglutination tubes containing
fresh medium were inoculated with the same cultures.
The first set served as controls under atmospheric
conditions, the second placed in Thunberg tubes and
evacuated, and the third set was placed in a dessicator
in the presence of alkaline pyrogallol and evacuated.
Similar staining procedures were followed as mentioned
above.
All smears were then examined microscopically.
55
Table V
Progressive Morphological Changes of Cultures
During Fermentation of Cellulose
Davs
Culture 1
Large free
spores 2 - i k ^
0
Few granular
cells and
terminal spores
Gram
rods
S x 0.5/^
Single and
short chains
Culture 2
Free
spores 1-^^"''
Resemble
coccoid forms
Few granular
cells
Stain unevenly
Gram 4- rods
6 x 0.
Single and
short chains
Culture 3
Free
spores 2-3,
Resemble
coccoid forms
Few granular
cells
8tain unevenly
Gram + rods
4- x 0.25^*'"'"
Single £Cnd
short chains
Gram variable
Gram variable
Gram variable
rods
rods
rods
2 Few cells with Few cells with Few cells with
granular ends
granular ends
granular ends
Few terminal
Few terminal
Few terminal
snores 2-3/^
snores 2_____ spores 2-k-s**'
Few gram
Few gram
Increase in
variable
rods
variable rods
terminal spores
Increase in
Increase in
3
and granular
terminal spores terminal spores
cells
and granular
and granular
Few free snores
cells
cells
4-
5
12
Increase in
Increase in
Incres.se in
terminal and
terminal and
terminal and
free snores
free spores
free spores
Few granular
Few granular
Few granular
cells
cells
cells
Increase in
Increase in
Increase in
free sporesfree spores
free sporesResembling
Resembling
Resembling
cdccoid forms
cdccoid forms
coccoid forms
Few terminal
Few terminal
Few terminal
spores and
spores
and
spores and
granular
cells
granular cells granular cells
Mostly coccoid- Mostly coccoid - Mostly coccoidlike spores
like spores
like spores
Few terminal
Few terminal
Few terminal
spores and
spores and
spores and
granular cell s granular cells
granular cells
Table V, coh't.
Davs
Culture 1
Practically all
lg
coccoid-like
months
snores
Culture 2
Culture ^
Practically all
coccoid-like
spores
Practically all
coccoid-like 4
spores
The progressive morphological changes of the
cultures were somewhat parallel. In each case, the
morphology of the inoculum was characterized by free
spores which resembled coccoid-like forms.
Five and
12 day old cultures had, in addition, a few terminal
spores and granular cells which were absent from the
IS month cultures.
In the early stages of fermentation, young
vegetative cells stained evenly and hence were readily
differentiated by the Gram reaction.
However, the
first apoea.ra.nce of granular cells did not respond
to Gram's or Kopeloff's method of staining.
The
ends of the cells stained dark with granules present
and faintly red in the centers.
The Fulton-Schaeffer
stain did not show the presence of spores.
After the
first day, the vegetative cells of culture 1 appeared
slightly curved and rounded at the ends and those
of cultures 2 and 3 were straight.
As the fermentation
57*
progressed, the granular cells became clearly
Gram negative.
At this stage granulation at the
ends was more evident and the first appearance of
oval terminal spores was observed.
The spores
were stained readily by the Fulton-Schaeffer method.
At the end of fermentation free spores were pre­
dominant and were stained easily.
In summation the morphological changes
consisted of the following sequences:
1- Vegetative rods which stained Gram positive
and occurred singly and in short chains.
2- Vegetative cells - Gram reaction was
variable.
Appearance of granular cells that stained
dark at ends and a few terminal spores.
3- An increased number of terminal spores
and granular cells together with a few free spores.
Gram negative cells were present.
4- An increased number of free and terminal
spores; few granular cells.
Free spores predominated which resembled
coccoid-like forms; decreased number of granular cells.
In the agglutination tube studies, the
morphological changes of the cultures during fermenta­
tion of cellulose were analogous to those described
nor was there marked difference in the chronological
sequence of morphological changes between semiaerobic and anaerobic conditions.
2— Morphology on Solid M e d i u m :
Suitable dilutions of cultures were prepared
with sterile water blanks and p lat ed out with dextrose
agar in Petri and Spray dishes for aerobic and
anaerobic conditions respectively.
Incubation was
at 370C and 60°C for 24- to 4-S hours.
this period,
stained.
At the end of
smears were made from the colonies and
At the same time fresh tubes of liquid
medium were inoculated from representative colonies
and incubated at 60°G.
Stains were made of 1 , 2 , and
6 day old cultures.
Dextrose agar slants were
inoculated from
colonies and incubated aerobically and anaerobically
at 60°C and 37°^ for 24- ~ ^
hours.
Similarly,
were made at 1 , 2 , and 6 day intervals.
stains
Transfers
were made from aerobic slants to new slants which
were then subjected to anaerobic conditions and vice
versa.
Incubation was at 37°^ and 60°C for 4-S hours
and stains made as mentioned above.
stains were made of 2 , 4, and
slants.
In addition,
week old culture on
All stains were examined microscopically.
59.
After
were
T he
24- h o u r s
similar under
at 60°C,
aerobic
following observations
Culture
forms.
Vegetative
All
The
were
cells were
more
curved
in size.
Cells
No spores
were present.
rods
occurred
Culture
phology
x 0 .5 ^."
singly.
less
of
than those
size.
from
No c h a i n s
and g r a n u l a r
al l
forms
cells were
singly and
4.0
somewhat
x
curved.
3
Gram positive
When
x O.^^^ln
2
which stained Gram negative;
occurred
6.0
and a few
ends were rounded.
Gram positive
3*0
rods
observed.
Culture
cells;
conditions.
were made.
vegetative
cells were
liquid medium.
or s p o r e s
a nd a n a e r o b i c
1
Gram positive
granular
growt h and mor pholog y
No
Cells
were
the
and granular
straight
and
present.
were made
sa m e
rods
mostly
spores were
transfers
each was
vegetative
to
slants,
the mor­
as d e s c r i b e d a b o v e ,
regard­
of o x y g e n t e n s i o n .
Growth
observed
first
characteristics
in t h e
after
cultures
i n c u b a t e d at
2 to 3 days.
of e a c h w e r e
3J°C w a s
Th e m o r p h o l o g i c a l
similar
to t h o s e
at
60°C
60 .
except that an increased number of granular cells
and a few terminal spores were noted.
In addition,
the vegetative and granular cells appeared to be
smaller.
Under reversed oxygen conditions,
morphology of each was again identical.
the
Six day
old cultures had Gram negative granular cells,
free and terminal spores.
Cultures 2 , U-, and S weeks
old showed predominantly free spores which resembled
coccoid-like forms.
Very few granular cells and
terminal spores were present.
Effect of Heat Treatment:
Morphological
Changes during Fermentation of C e l l u l o s e :
The cultures were heated at 100 — 1 05°C for
6 hours.
Fresh tubes of medium were then inoculated
with 1 ml.
days.
of inoculum and incubated at 60°C for 5
Stains were made of the original cultures
and daily thereafter during the progress of fermen­
tation.
After the fermentation was complete the
process was repeated.
The cultures were kept in an
ice box for two months and again treated as above.
The cultures when previously heated at
100 - I O5 0 C did not show any significant difference
in morphological changes during the progress of cel­
lulose fermentation.
Hoxvever, since fermentation was
6l.
e n de d in 5 to 6 days,
at the
time
a slight d i f f e r e n c e w a s 'n o t e d
spores occurred-
T h e i r presence w a s
o b s e r v e d 1 to 2 days l a t e r than in the u n h e a t e d
cultures.
R e p e a t e d h e a t i n g d id no t alter an y m o r ­
phological
changes d u r i n g f er mentation.
m o n t h o ld cultures e x h i b i t e d the
The two
same c h a r a c t e r i s t i c s .
A l t h o u g h h e a t i n g d e l a y e d the a p p e a r a n c e of
the cycli c
phases of e a c h culture,
the c h r o n o l o g i c a l
order of the m o r p h o l o g i c a l changes was not al tered*
Subsequent
transfers o f h e a t - t r e a t e d cultures f u l l y
r e s p o r e d their usual m o r p h o lo gy .
ment p r e c l u d e s
As the heat
the p o s s i b i l i t y of n o n — spore f o r m i n g
o r g a n i s m s b e i n g present,
the a p p e a r a n c e of G r a m
n e g a t i v e g r a n u l a r cells du ri n g the
f e r m e n t a t i o n indicates
the life
t r ea t­
la.ter stages
of
that they are m e r e l y p a r t of
c ycle of the G r a m p o s i t i v e
spore-forming
rod.
D- B I O C H E M I C A L REACTIONS;
1• M e d i a :
V a r i o u s sugars,
saccarides,
glucosides
n u t r i e n t b r o t h base),
n u t r i e n t broth,
were
p o l y h y d r o x y alcohols,
(0.5 per cent sugars,
n i t r a t e broth,
etc.
litmus milk,
d e x t r o s e agar slants,
i n o c u l a t e d w i t h e a ch culture.
poly—
in
gelatin,
and p o t a t o agar slar
I n c u b a t i o n was at 6 0 °C
62 *
and 37°C for two weeks.
2. Motilitv:
Motility was determined during the progress
of cellulose fermentation by use of a. hanging drop
on a warmed stage.
3• Flagella Stains:
The Gray technique (4-0) was used in this
procedure.
Dextrose agar slants were inoculated with
each culture and incubated aerobically for 2k- to k-B
hours at 60°C.
Loop transfers were made to 10 ml.
sterile distilled water blanks which were then incubated
at 60°C for 24- hours.
At the end of this period,
smears
were made on clean slides and allowed to dry.
A raordant*was applied to the smears for 20
minutes and then washed with distilled water.
Basic
fuchsin stain-** was added for 15 - 20 minutes and
followed by tap water.
The slides were air dried
without blotting and observed under a microscope.
Liquid cellulose medium was inoculated from each slant
for confirmation of cellulose fermentation.
* Potassium alum saturated aqueous solution
Mercuric chloride saturated aqueous solution
Tannic acid 20 per cent aqueous solution
5 ml.
2 ml.
2 ml.
*"*0.4- ml. of saturated alcoholic solution of basic
fuchsin was added to 9 ml* of mordant and filtered.
63
Table VI
Biochemical Activities of Cellulose Fermenting
Cultures at 60°C.
Medium
containing:
Nutrient
Broth
control
Culture 1
Culture 2
Culture S
Turbid
Turbid
Turbid
Sucrose
Acid
Acid
Ac id
Maltose
Acid
Acid
Acid
Lactose
Alkaline
Alkaline
Alkaline
Dextrose
Levulose
Galactose
Cellobiose
Salicin
Acid
Acid
Acid
Slightly acid Slightly acid Slightly acid
2 weeks
2 weeks
2 weeks
Slightly
Slightly
Siightly
Alkaline
Alkaline
Alkaline
Acid
Mannitol
Acid
Alkaline then
Acid after
^ da vs
Nitrate
No Reduction
Acid
Acid
Acid
Acid
Acid
Acid
No Reduction
No Reduction
Litmus Milk
No Chance
No Change
No Change
Haffinose
Alkaline
Alkaline
Alkaline
Dextrin
Acid
Slightly
Acid
Acid
Slightly
Acid
Acid
Slightly
Acid
Acid
Ac id
Acid
Slightly
Acid
Acid
Slightly
Acid
Acid
Slightly
Acid
No Change
Slightly
Acid
No Change
Slightly
Acid
Adonitol
1-arabinose
d—mannose
Dulcitol
Gelatine
Mellizitose
No Chanee
No Chance
Slightly
Acid
Table VI, con't
Medium
containing
Culture 1
Culture 2
Culture "5
No Change
Slightly
Acid
Slightly
Acid
No Change
Slightly
Acid
Slightly
Acid
No Chanse
Slightly
Acid
Slightly
Acid
Acid
Acid
Acid
Hydrolysis
Moist
transparent
beaded
filiform
growth
Hydrolysis
Moist
transparent
beaded
filiform
growth
Hydrolysis
Moist
transparent
filiform
growth
Inositol
d-me thyl
fflucoside
Amverdalin
Starch
Starch ae:ar
Potato
Dextrose
agar
Dextrose
aear
-ditto-
-ditto-
-ditto-
According to the biochemical activities
recorded in 'Table VI, the cultures varied only to a
slight degree.
With mannitol culture 1 produced an
alkaline reaction for 4- days and an acid reaction
thereafter.
Cultures 2 and 3 developed strong acidity
in 24- hours.
Although slight variations were noted in
acidity, this difference was not significant.
At 60°C
the reactions were generally evident after 3 days and
at 37°C after 6 to 3 days.
Gas was absent in all
fermentations•
All cultures were actively motile after 24
and 43 hours.
cultures.
Flagella were peritrichous in all three
Those of culture 1 were longest, varying from
65.
g - 10 times the size of the cells and those of cultures
2 and 3 were 5 "* ^ and- 4- — 6 times, respectively.
It is interesting to note that the reactions
of galactose, lactose, and raffinose were aklaline
whereas most of the other carbohydrates were acid in
reaction.
Since galactose is a constituent of lactose
and raffinose, it is possible that the alkaline reaction
may be attributed to some reaction involving the
galactose molecule.
,
66
E- FERMENTATION OF OTHER CELLULOSIO MATERIALS:
Media:
The Vilj oen, Fred and Peterson medium (B-l)
was used as a base.
Control without cellulose, cotton,
white pine wood, filter paper plus white pine wood,
excelsior, filter paper plus excelsior, cacao shells,
filter pacer plus cacao shells, towel paper, filter
paper plus towel paper, and filter paper were used
separately in each medium.
One ml. of inoculum was
used for each tube of medium.
60°C for 2 weeks.
Incubation was at
67
Table VII
Fermentation of Cellulosic Materials
Medium A
containing
Control
without
cellulose
Filter
Paper
Culture 1
Slight
growth
Culture 2
Slight
growth
Culture 1
Slight
growth
Complete in
4— 5 days
Complete in
4— 5 days
Complete in
4— 5 da,ys
Cotton
Complete in
5-6 days
Complete in
6-7 days
Complete in
6-7 days
Towel
Paper
Partial
Partial
Partial
decomposition decomposition decomposition
after 14- days after 14- days after l4 days
White
pine
wood
Growth but
no visible
decomposition
after 14- days
Growth but
no visible
decomposition
after l4- days
Growth but
no visible
decomposition
after l4 days
White pine
wood plus
filter
paper
Paper
decomposed in
4— 5 days.Wood
not attached
after 14 days
Paper
decomposed in
$-6 days.Wood
not attacked
after 14 days
Paper
decomposed in
6-7 days.Wood
not attacked
after 14- days
Excelsior
Ho growth or Ho growth or Ho growth or
decomposition decomposition decomposition
after 14 days after 14- days after l4- days
Excelsior
plus
filter
paper
Paper
decomposed in
5-6 days.Wood
not attacked
after 14- days
Cacao
shells
Slight growth Slight growth Slight growth
but no
but no
but no
visible
visible
visible
decomposition decomposition decomnosition
after 14- days after 14 days after 14 days
Cacao
shells
plus
filter
paoer
Paper
decomposed in
6-7 days.Wood
not attacked
after 14- days
Paper
decomposed in
5-6 days.Wood
not attacked
after 14 days
Paper
Paper
Paper
decomposed in decomposed in decomposed in
7— S days
6-7 days
5-6 days
shells not
shells not
shells not
visibly
visibly
visibly
decomposed
decomposed
decomposed
6S.
The rate and extent of fermentation varied
with each type of cellulosic substance.
Free cellulose
was attacked readily whereas woody materials were very
resistant to decomposition.
The addition of the woody
ma.terials to filter paper had no significant deterring
effect on the breakdown of free cellulose.
other hand,
On the
the presence of free cellulose in the
medium containing woody materials did not aid in
initiating the breakdown of the latter substance.
Although towel paper was partially decomposed the
fermentation was sluggish and did not resemble the
normal activity on filter paper.
Although slight variations of the rate of
fermentation of filter paper in the presence of
excelsior,
pine wood or cacao shells were noted,
there
was no sharp distinction observed between the fermen­
tation of various forms of cellulose.
results were based only on observation,
Since these
the inhibitory
effects of lignin associated with cellulose were in
agreement with the work of Olson and his coworkers (2S)
who reported that fermentation of free cellulose was
not inhibited by the presence of cellulose-lignin
materials.
However, no attempts were made to confirm
their work with regard to the effect of lignin content
of cellulose on the rate and extent of fermentation.
69
F- ANALYSIS OF FERMENTATION PRODUCTS:
In the study of the end-products of cellulose
decomposition, 2 liters of medium B-l plus 20 grams of
filter paper were used except for gas production in
which half of this amount was used.
Incubation was
at 60°C until the end of fermentation.
1- Gas analysis;
One liter of sterile medium was inoculated
with 25 ml* of culture.
The flask was placed at 60°0
and sealed with a rubber stopper having an inlet and
an outlet.
The inlet was used for flushing the system
with nitrogen gas previously passed through alkaline
pyrogallol solution and the outlet was connected to
a train of gas absorbers and a graduated liter burette.
Concentrated sulfuric acid was used for drying, freshly
prepared and dried cupric phosphate for hydrogen
sulfide absorption,and 4-0 per cent potassium hydroxide
for carbon dioxide absorption.
The burette contained
saturated sodium chloride solution plus a little
concentrated hydrochloric acid and methyl red indicator
over whdch hydrogen, methane, and nitrogen were collected.
At the start of the fermentation the system was
flushed with nitrogen gas for one half hour.
Gas volumes
70*
were recorded each day.
Each daily gas sample was
collected over saturated sodium chloride solution
and subsequently analyzed in an Orsat Gas Apparatus (^l).
Hydrogen and methane were determined by this procedure
and calculated to standard conditions.
.
71
Table V I I I •
Average Volume of Gas Produced
in Cellulose Fermentation
No.of
RepliCulture cates
Ml.gas produced
by days
1
2
1
4
Total
Volume
gas
produced
ml.
Percent Volume
gas produced
by days
1
2
1
4
1*
6
317
225
-
1290
24.5 57.9 17.6
2
5
147 543 112
-
£02
10.3 67.7 14.0
3
6
10^ 431 30g 119
966
11.2 44.6 31.9 11.:
-
* Culture No.l - produced an average of 7 ml. hydrogen
sulfide during fermentation;
this has not been included
in the volume of gas in the above table.
did not produce hydrogen sulfide.
Cultures 2 and 3
Table IX
Average Per cent of Each Gras Produced
in Fermentation of Cellulose
No.of
Culture Repli­
cates
Per cent produced by Days
h2
1
co2
C%
1
6
54.1 ^5-9 -
2
5
69.4 22.0
3
6
62.3
i2
2
C02
25.0 75.O
0%
-
H2
3
C02
16.6 61.2
CHl|
4h2 co2 c%
-
6.6 36.0 63.6 0 .4- 4-.9 95.1 -
24.9 12.S 46.1 4-5.9 3-0 30.0 6S.4 1.6 24-.3 7^.9
Per cent of
Total Gras
h2 C02 CHj^
-
31.0 69.O
-
37.2 60.3 1.9
0.6 40.1 54.3
-
5.6
73.
70
-
<50
-
A
60
S/a if3d JtV/7 703
Gas Pr o c /u c f i o n
F "- t- m e.ntat / on
HO
—
dA/dOddd
C L A /tu n c
C
-
lo —
/■
C**/tuf-c 2..
30 -
20
/
DAYS
FIGURE I
i
D u rin g
it*utr~C
3.
7^
The average constituent analysis of gases
produced during fermentation of cellulose are rep­
resented in Tables VIII and IX and Figure 1.
In
general, maximum yields were obtained during the
second day of fermentation.
gas varied significantly;
3 intermediate,
The total volume of
culture 1 being highest,
and 2 lowest.
also were different.
The daily percentages
Only culture 3 produced any
gas after 3 days.
After one day of fermentation, the yield
of hydrogen was higher than carbon dioxide.
On the
second day the carbon dioxide was predominant and
progressively increased in volume throughout the
fermentation while hydrogen decreased steadily after
the first day.
In both cases, where methane was
produced it steadily decreased until the end of
fermentation.
The probable reason for the low carbon
dioxide— hydrogen ratio at the beginning may be the
result of a high initial pH in the medium,
tending to absorb some carbon dioxide.
thus
As the
acidity increased, less carbon dioxide was absorbed.
Also at this point,
the products formed were probably
more susceptible to oxidation.
As a result of these
75two factors, the carbon dioxide-hydrogen ratio
became increasingly more in favor of carbon dioxide*
Although results show that the yields of
hydrogen and methane were highest and carbon dioxide
was lowest at the beginning of fermentation and at
the end the reverse was true, experimental evidence
as to the sources of these gases and the probable
role they play in cellulose breakdown is lacking.
As early as 12>75> ^opoff (^2a) considered that
cellulose was probably a precursor of methane and
hydrogen.
Hoppe-Seyler (M-2b) deduced that cellulose
was broken down according to the equations:
^G 6H 10° 5 ^n *•* n h 2° = n ^c 6H 12° 6 ^
(C6H12°6 )
s
3gg2
Acetic acid was suggested as a possible intermediate
product.
Later he showed that methane occurred in the
fermentation of calcium acetate.
In similar circum­
stances, Coolhaas (*i2c) obtained methane from calcium
salts of acids such as propionic, gluconic, and lactic.
Sohngen (h2d) produced methane from calcium salts of
formic and butyric acids.
The presence of hydrogen as an end-product is
typical of anaerobic fermentations and indicates a
76.
similarity of the thermophilic fermentation of
cellulose to carbohydrate breakdown by other an­
aerobic spore-formers in so far as gas production
is concerned.
2- Chemical Analysis:
Preparation of Fermented Liquor
The fermentation flasks were removed from
the incubator and immediately cooled to room temper­
ature with tap water.
The liquor was siphoned through
a cheese cloth into a flask immersed in ice water.
Preparation of Residual Cellulose
To the flask containing residual cellulose,
200 ml. of 1-5 dilute hydrochloric acid were added,
>
stirred and allowed to stand one hour.
The residue
was then filtered in a Buchner funnel containing a
known weight of filter paper and washed with one
per cent potassium hydroxide solution and distilled
water.
The residual cellulose was spread over the
surface of a large dish and placed in a 100 - 105°0
oven until a constant weight was obtained.
a- Determination of titratable acidity.
Fifty ml. aliquots of liquor were titrated
with standard sodium hydroxide solution before and
after fermentation.
The difference was calculated as
titratable acidity.
b- Determination of reducing sugars.
Aliquots of liquor were employed for the
determination of reducing compounds (calculated as
glucose) before and after fermentation.
The micro
method of Stiles, Peterson, and Fred (^3 ) w a s used
in this procedure.
c- Determination of volatile solvents.
An aliquot of liquor was made slightly
alkaline to litmus paper.
The volatile solvents
were then separated from the remainder of the mix­
ture by distillation.
The residual solution was
retained for determination of volatile acids and
lactic acid.
The determination of the volatile neutral
compounds was made by the method of Stahly, Osburn,
a.nd sVerkman (MO.
An aliquot of neutral volatile
distillate was oxidized by potassium dichromatephosphoric acid mixture to the corresponding acids
and then followed by distillation.
Aliquots of the
distillate were titrated and partitioned with
anhydrous ethyl ether according to the procedure of
Yi/'erkman (M?).
Acetone was determined by titration
of an unoxidized aliquot with iodine in alkaline
solution according to Goodwin's method (MS).
7S.
d- Determination of volatile acids.
The residual mixture from the neutral solvents
was adjusted to pH 3.0 with concentrated sulfuric acid,
using congo red as an indicator, and steam distilled.
Volatile acids were determined by the partition method
of Osburn, Wood, and Werkman
determination
is based on the difference in titratable acid of the
distillate before and after partition with anhydrous
ethyl ether.
e- Determination of lactic acid.
The method of Friedmann and Graesar (^-SO was
employed.
The lactic acid was oxidised to acetalde-
hyde by potassium permanganate in the presence of
manganese sulfate-phosphoric acid mixture.
The
aldehyde was distilled into cold strong sodium
bisulfite solution and titrated with iodine according
to the method of Clausen (^4-9)*
f- Determination of fixed acids.
The titratable value of fixed acids was cal­
culated as succinic acid by subtracting the values of
unfermented liquors plus volatile acids and lactic acid
from the value of titrated fermented liquor.
Table X
CT\
t"~-
Average Analysis of Fermentation of Cellulose under Nitrogen Gas
(Products given in per cent of Cellulose Fermented)
Reducing
Non­
Cellu­
Buty­
volatile
Sugars
lose
No. of
as
Lactic Acid as Carbon
Ethyl Acetic ric
Carbon
Cul- Repli- FerAcid Succinic Dioxide Methane Recovered
ture cates mented Alcohol Acid Acid Glucose
.
$
._
..
1°
1
6
70.0
13.6
19.7
2
5
35.0
13.5
19.2
3
6
if-6.0
1*1.7
22.5
A ■
..A.
i
5.6
. ..... i .
p
d;q
dq
37*2
1.1
0.2
15.3
-
*12.*1
0.9
0.2
16.7
0.5
92.7
—
33.1
0.7
0.3
13.?
0.1
as.i
-
92.2
Table XI
Average Analysis of Fermentation of Cellulose under Atmospheric Conditions
160
(Products given in per cent of Cellulose Fermented)
Reducing
Non­
Sugars
volatile
No.of
Lactic Acid as
as
Carbon
Cul­ Repli- Cellulose Ethyl Acetic Butyric
Acid
Acid
Acid
Glucose
cates
Fermented
Alcohol
Succinic
Acetone
Recovered
ture
s
1o
%
1o
1
ill-
62.5 .
12.6
20.4
2
14
54.0
13.2
20.6
3
12
in.7
15.3
23.1
%
7.0
. A _____
djO
d!0
*
70
31.*
1.20
0.25
5.2
77.2
-
3*. 7
1.10
0.30
1.6
75. &
-
37.7
0.77
0.30
0.5
77.4
Table X gives the avera.ge analysis of end-products
produced from fermentation of cellulose under nitrogen
gas.
It is noted that culture 1 produced the highest
per cent decomposition of cellulose and culture 2
the lowest.
Although the per cent of ethyl alcohol
was slightly higher in case of fermentation 3 > "the
relative amounts were within a narrow range from 13»5
to 14-.7 .
However,
the amount of volatile acids was
significantly different.
Culture 1 produced about
25 per cent volatile acids of w h i c h 5.7 per cent was
butyric.
3 to 1.
The ratio of these acids was approximately
Cultures 2 and 3 produced only acetic acid
and in lower yields of total volatile acid content
than that of culture 1.
The amount of reducing
sugars was nearly the same with cultures 1 and 3 an&
slightly higher with culture 2.
Somewhat parallel
results by cultures 1 and 2 are indicated in yields
of lactic acid and carbon dioxide.
However,
latter culture produced methane in addition.
3 gave lower yields of these substances.
the
Culture
The balance
of products from each fermentation when compared to
the fermented cellulose showed that from & to 12 per
cent of carbon is unaccountable.
Some of this loss
may be attributed to the gummy-lilce material which was
32.
soluble in acid and consequently lost in the preparation
of the residual cellulose.
Other unaccounted carbon
may be due to calculation of total reducing substances
as glucose and non-volatile acid as succinic acid.
There is also the possibility of unknown intermediates
which may add to the difference of carbon balance.
As indicated in table XI,
the amount of cellulose
fermented under atmospheric conditions as compared to
that under nitrogen gas was lower with cultures 1 and
3 and higher with culture 2.
Apparently the presence
of oxygen favors the decomposition of cellulose by
culture 2.
The relative yields of ethyl alcohol and
volatile acids were approximately the same under both
conditions.
However,
lower in each case.
the reducing sugar content was
Ths most significant drop was
observed with culture 2.
The yields of lactic and
succinic acids were about the same as under nitrogen
gas.
Acetone was found to be highest with culture 1
and lowest with culture 3*
^He fact that gases were
not determined gives evidence of the lower carbon
recovery as compared to that under nitrogen gas.
3“ Qualitative Chemical Analysis:
a- Acetyl methyl carbinol
Deep tubes of medium were inoculated and
incubated at 60°C for 6 days.
Each day 15 ml. of fermenting liquor were
pipetted into 3 test tubes in equal proportions.
each of these 5
To
of 0*25 per cent creatine solution
in 4-0 per cent potassium hydroxide were added and then
the tubes were incubated at 37 >
£>0°C for 4-25
hours.
According to the method of O'meara (5 0) a
positive test for acetyl methyl carbinol gives a red
color.
An acetyl methyl carbinol dilution of I-25OOO
indicates a positive test and at l-500°0 negative.
In every test, acetyl methyl carbinol was
absent.
It is suggested that the mechanism of the
fermentation does not involve a benzoin type of
condensation in which two molecules of acetaldehyde
condense through the carbonyl linkages.
b- G-lucososazone
A volume of fermented liquor was deproteinized
by basic lead acetate followed with potassium oxalate
and then concentrated under vacuo at 4-5°Q*
An osazone
8&.
was then prepared with phenylhydrazine solution and
examined microscopically.
A similar derivative of
glucose was prepared and compared with it.
Mixed
and individual melting points were taken.
Microscopic examination of known and unknown
osazones indicated similarities with each respective
culture.
The melting points also revealed that the
derivatives of glucose were identical to those of
each culture.
Mixed melting point readings were
correspondingly similar.
The recorded melting point
of glucosazone in the literature is 205°C.
Hence,
it
is suggested that each culture produces glucose as an
intermediate and end-product.
Table XII
Melting Points of Osazones
Test
m.n . °C
Culture 1
203
-
206
Culture 1
4erlucosazone
203
-
205
Culture 2
203
-
205
Culture 2
4elucosazone
203
-
206
Culture 3
202. 5-205.5
Culture 3
4s’lucosazone
203
-
205
G-lucosazone
203
-
205
£56.
G- EFFECT OF TOLUENE AND SODIUM FLUORIDE ON THE ENDPRODUCTS OF CELLULOSE FERMENTATION:
Despite the amount of research on the thermo­
philic fermentation of cellulose,
there is very little
information on the intermediary mechanism of cellulose
breakdown.
As the use of toluene and sodium fluoride
have cast light on the fermentation mechanism of
glucose dissimilation by yeast and bacteria,
the
effect of these compounds was studied in relation to
the thermophilic cellulose fermenters.
1- Effect of Toluene:
In order to determine the effect of toluene
concentration on the rate of cellulose breakdown,
large test tubes containing medium and 0.5, 1.0, and
1.5 per cent toluene were inoculated with 1 ml.
culture and incubated at 60°C for 10 days.
of
27<
Table XIII
Effect of Toluene on Fermentation
of Cellulose
i
(Time Necessary to C o m p l e t e •Fermentation)
Culture 1
Culture 2
Culture 3
Medium
Da vs
Davs
Davs
Control
k- - 5
* - 5
14- - 5
0.5 $>
toluene
6 - a
6 - a
6 - a
1 .0#
toluene
10*
10*
10*
1 .5#
toluene
1 0**
1 0 **
1 0 **
*
as G-ood growth,
slight decomposition of cellulose
** = Very slight growth, no decomposition of cellulose
gg.
As indicated in table XIII,
the inhibitory
effect of toluene on the rate of fermentation increased
as the concentration of the toluene was increased.
Although the fermentation with O.R per cent toluene was
complete in 6 to S days,
the amount of decomposed
cellulose was significantly smaller as compared with
that of controls.
The amount of cellulose fermented
was still smaller with 1.0 per cent toluene,
and no
breakdown of cellulose took place in 1.5 per cent
concentrat ion.
In another series of experiments large scale
fermentations were run in the presence of O .5 per cent
toluene.
Two liters of medium containing 0.5 per cent
toluene were inoculated and incubated at 60°C for 6
days.
Chemical analysis of end-products was deter­
mined according to the methods outlined under section F.
It is noted in table XIV that in each case
the per cent breakdown of cellulose was lower when
compared to fermentations conducted in the absence of
toluene
(Tables X and X I ).
The decrease in rate of
each fermentation was correspondingly analogous.
general,
In
the yields of neutral volatile compounds,
volatile acids, and lactic acid were markedly decreased.
Table XIV
The Effect of 0*5 per cent Toluene on Fermentation of Cellulose
£
to
(Average Analysis based on per cent Cellulose Fermented)
Reducing
Non-volSugars
atile Acid
No. of
Carbon
Repli­ Cellulose Ethyl Acetic Butyric Calculated Lactic Calculated
ReAcid
Acid
Fermented
Alcohol
as glucose Acid as Succinic Acetone covere(
Culture cates
At
fi
(<o
10
1
5
^3-7
2
5
23.2
6 A
12.5
3
5
25.2
9A
1S .3
%
t*
%
-:c
%
22.7
J+s.5
0.6
0.1
o.£T
Si.5
-
5^.0
oJt
0.2
o A
73.s
Ij-S.l
0 A
0.1
0.02
76.6
90.
It is interesting to note that the ratio of acetic
acid to butyric acid is approximately 1 to 2 , whereas
without toluene it is 3 to
Another striking dif­
ference was found in.per cent of reducing sugars.
With toluene much higher yields were obtained ranging
from 12 to 17 per cent increase.
The per cent carbon
recovery was practically parallel to those without
toluene.
A third series of experiments was conducted
to study the effect of toluene when added after three
days of normal fermentation.
Two liters of medium
were inoculated and incubated at 6 0 °C.
three days,
35^ m ^*
At the end of
liquor were removed and enough
toluene was added to the remaining liquor to bring its
concentration to 1.5 per cent.
incubated at 60°G.
The flasks were again
Similar aliquots were removed
after the H-th and 5th days of fermentation.
Each
sample of liquor was analyzed for volatile acids,
ethyl alcohol, and reducing sugars.
Residual cellulose
was determined at the end of the experiment.
The data in table XV show the effect of
toluene when added after three days of normal fermen­
tation.
In each case the most significant change was
noted in the rapid accumulation of reducing sugars.
Table XV
Effect of 1.5 per cent Toluene added after 3 days of Fermentation
Aliouots*
A
B
c
No. of
Replicates
if
if
4
Product
Butyric Acid
Acetic Acid
Ethvl alcohol
Glucose
Butyric Acid
Acetic Acid
Ethvl alcohol
Glucose
Butyric Acid
Acetic Acid
Ethyl alcohol
Glucose
Cellulose
fermented
fo
Culture 1
Culture 2
gms./liter gms./liter
liouor
liouor
.430
0.667
I.13.£
0.346
... 0.324
1.007
1.10'S
—
.....
1.007
_
0.S4S
0.3oS
2.000
0.223
1.113
- l.b4s
°»-5P-9
—
Culture 3
gms./liter
liauor
—
.. _0_.9S3
o.4so
0.290
1.2S0
0.440
1.23S
—
0.370
2.S42
0,211
0.301
2.420
1.22S
0.4SS
1.9S6
22.1
2S.0
20.2
0.622
A = Aliquot of liquor after 3 day fermentation
before toluene was added.
B « Aliquot of liquor after 4- day fermentation
in presence of toluene for 1 day.
0 = Aliquot of liquor after 5 day fermentation
in presence of toluene for 2 days.
92.
After the first day in toluene the increase of sugars
was approximately 100,
5 0 » a-nd 25 per cent by cultures
1, 2, and 3> respectively.
At the end the increase
rose to as high as 150 per cent.
Ethyl alcohol
remained practically constant with exception of
slight increases by cultures 1 and
^he total
volatile acid content increased about 25 per cent
in each case after the first day in toluene and then
decreased slightly.
A remarkable change of acetic-
butyric acid ra.tio was observed.
the ratio was approximately
At the beginning
to 1 and 1 to 2 at
the
end.
'
To assign any particular role in altering
the course of normal reactions to toluene would be
highly speculative.
However,
in view of the evidence
presented above, the following factors are worthy of
consideration.
The rapid accumulation of sugars and
simultaneous decrease of neutral volatile compounds
and volatile acids suggest that the inhibiting effect
of toluene takes place at the stage of glucose break­
down.
These results corroborate those of Pringsheim (ll,12)
and Woodman and Stewart
(1 5 ,1 6 ).
However,
it is apparent
that toluene at a higher concentration inhibits the first
stage of cellulose breakdown since,
according to table XV,
93it appears that fermentation was arrested once an
equilibrium was established*
2— Effect of Sodium F l u o r i d e :
The initial investigation concerning the
effect of fluoride was essentially devoted to determin­
ation of the molar concentration w hich still allowed
cellulose to be attacked.
The second phase was
centered in a study of physiological adaptation of
the cultures to ferment cellulose in a medium contain­
ing fluoride.
The last phase was the analysis of end-
products from fermentation in the presence of fluoride.
Large test tubes containing medium and sodium
fluoride varying from 0.0^- M to 0.002 M concentrations
were inoculated with 1 ml. of culture and incubated for
10 days at 60 °C.
For physiological adaptation studies,
cultures with and without decomposition of cellulose
were transferred to the media mentioned above.
process was repeated.
This
(As indicated in table XVI
0.005 M cultures which showed no decomposition of
cellulose and O.OO25 M cultures w h ich fermented cel­
lulose were used.)
9 4-.
Table XVI
Effect of Fluoride Concentration on the Rate
of Fermentation of Cellulose
(Time Necessary to Complete Fermentation)
Concentration
of
Sodium
Fluoride
Culture 1
Davs
Culture 2
Culture 3
Davs
Davs
.OU-M
10 Negative
10 Negative
10 Negative
•02M
10 Negative
10 Negative
10 Negative
•OlM
10 *
10 *
10 *
10 **
10 **
10 **
.OO33M
o_o * * *
10 * * *
.OO25M
6 - g
6 - 7
7 - 9
.002M
5 - 7
5 - 6
6 - 6
Control
4- - 5
14. _
4- - 5
.OO5M
5
* — Slight growth
** = Good growth, no cellulose breakdown
*** _ Slight decomposition of cellulose
***
95Table XVII
Physiological Adaptation of Organisms
to Fluoride in Fermentation of Cellulose
after two Successive Transfers
(Time Necessary to Complete Fermentation)
Concentration
of
Sodium
Fluoride
•02M
Culture 1
Culture 2
Davs
Davs
(.OO5M NaF Cultures)
Culture 5
Davs
10 Negative
10 Negative
10 Negative
. .01M
10 *
10 *
10 *
•OORM
10 **
S - 10
.0071M
10 ***
7 - 9
& - 10
.0025&£
7 - 9
S — 6
6 - 7
-
ip _ g
ip _ g
•002M
Control
5
6
10 ***
- 5
....> - 5 ____ .. .K - 5
(•OO25M Cultures)
.02M
10 Negative
10 Negative
10 Negative
•OlM
10 *
10 *
10 *
10 **
-j_q
,OORM
.0077M
3
LT
C\J
•!
0
0
.002M
Control
***
-J^Q
***
& - 10
...6 - .7..
& - 10
5 - 6
> - b .
^ - 5....
^ - 5_____
6 - 7
h -
ip _ 5
5 - 6
ip _ c;
* = Slight growth; no cellulose breakdown
** = Slight decomposition of cellulose
*** = Incomplete fermentation
The data pertaining to the effect of fluoride
concentration on the rate of cellulose “
b reakdown are
presented in table XVI.
It is evident that the
organisms are quite sensitive to fluoride since they
attack cellulose at relatively low molar concentrations.
The first sign of cellulose breakdown was observed at
.003M concentration.
At this stage fermentation appeared
sluggish and incomplete.
However,
as the concentration
of fluoride was decreased there was a corresponding
increase in rate of cellulose breakdown.
Although the
fermentations at .OO25M and .002M fluoride concentrations
were complete the amount of cellulose decomposed was
markedly smaller than that of the controls*
Only
growth was observed in .00511 and .01M concentrations
and no growth at .02M.
The apparent low tolerance
of these cultures when compared to other organisms
which can grow in fluoride as low as .04-M concentrations
at 30°C suggests that the high temperature of incubation
may increase sensitivity to fluoride.
The question of physiological adaptation to
fluoride was particularly emphasized by Meyerhof and
Kiessling
(5 1 ,5 2 ).
Their findings revealed that the
inhibitory effect of sodium fluoride is greatest at
the beginning of fermentation and is gradually overcome
as the reaction proceeds.
Therefore,
it would be
logical to assume that if an organism was exposed to
fluoride for a longer period of time,
function
the physiological
would tend to adapt itself to the particular
environment.
Table XVII shows the results of only
two successive transfers.
It is noted that at .OO5M
concentration slight decomposition of cellulose took
place whereas in the preliminary tests no breakdown
of cellulose occurred.
At other concentrations,
there
was some evidence of adaptation as the period of
fermentation was decreased.
A concentration of .01M
gave growth but no decomposition of cellulose.
Culture 2
responded more effectively than the other cultures.
Although the results are not entirely conclusive,
they
tend to support the contention that a physiological
adaptation at lower levels of fluoride concentration is
poss ible•
To study the effect of fluoride on the endproducts of fermentation,
2 liters of medium containing
.002M sodium fluoride were inoculated and incubated at
60°C for 6 days.
Chemical analyses on the liquor were
performed according to the methods outlined in Section F.
The results representing the effect of .002M
fluoride on the end-products of fermentation of cellulose
are given in table XVIII.
In each case, the amount of
Table XVIII
#
Average Analysis of Fermentation of Cellulose
160
in Presence of .00211 Sodium Fluoride
(Products Criven in Per cent Cellulose Fermented)
Reducing
Non-Volatile
No.of
Sugars
Acid
Carbon
Repli- Cellulose Ethyl Acetic Butyric calculated Lactic calculated
ReCulture cates Fermented Alcohol Acid
Acid
as glucose Acid as succinic Acetone covered
75
to
'0
70
p
.P
... p
....
.
1
2
29.2
g.lj-
13.6
2
2
2S .0
6.9
9.0
3
2
32.3
9.5
19.6
9.1
21.1
5.S
o .k
2.8
6O .3
.-
22.2
l.l
0.2
1.9
67,0
2S .1
'O A
0.2
0.3
-
99.
cellulose fermented was considerably less than under
nitrogen,
atmospheric conditions,
X, XI, XIV).
or toluene (tables
This decrease ranged from 15 to 60. per cent,
and it is obvious that the organisms are extremely
sensitive to fluoride.
In general,
reducing sugars,
ethyl alcohol, and volatile acids were lower in varying
degrees.
Significantly enough the acetic-butyric acid
ratio approaches 1 whereas in normal fermentation it
is 3 to 1 and with toluene 1 to 2.
While lactic acid
remained fairly constant with cultures. 2 and 3> a
marked rise from 1 to 5 *^ P er cent was observed with
culture 1*
On examination of the data,
it is noted
that the carbon recovery is lower than that obtained
in previous experiments
(tables X, XI, XIV).
carbon dioxide was not determined.,
Since
it is likely that
this compound may be responsible for some of the lost
carbon.
Evid.ence concerning the actual function of
fluoride in thermophilic fermentation of cellulose is
very scant.
A perusal of the literature does not reveal
any information relative to the mechanism of cellulose
breakdown.
Hence,
it would be merely supposition to
suggest a possible mechanism based on our present know­
ledge.
However,
there are some points worthy of note
lOOi
The fact the.t the organisms are highly
sensitive to fluoride may be indicative of a mechanism
such as proposed by Meyerhof for yeasts, however,
it
should not be overlooked that the toxic effect of
fluoride is undoubtedly increased at higher temperatures.
Second,
the low per cent of sugar suggests
it is unlikely that fluoride specifically inhibits any
important step in the dissimulation of glucose.
The
inhibition of fluoride in this case may be due simply
to a general poisoning effect on the cell.
;
H- Experiments on Fixation of Acetaldehyde bv the
Sulfite Method*
As in previous experiments with toluene and
fluoride,
the initial phase of this work was confined
to a study of the effect of concentration of sulfite
on the rate of fermentation*
Deep tubes of medium containing from 0*5 to
2*0 per cent sodium sulfite were inoculated with
1 ml. of culture and incubated at 60°C for 10 days.
In another series normal fermentation was allowed to
proceed for 3 days and then enough sodium sulfite
was added to bring its concentration in the range
stated above.
The tubes were incubated for 7
additional days.
The results presented in table XIX show the
effect of sulfite concentration on the rate of cel­
lulose breakdown.
stopped growth,
Since more than 0.5 P er cent
it is apparent that sulfite is ex­
tremely toxic to these organisms.
The fact that
researches have been conducted with various organisms
with as high as 3 per cent sulfite lends support to
this observation.
However,
it must be realized also
that the inhibitory effect of sulfite is more pronounced
Table XIX
Effect of Sulfite Concentration
on Fermentation of Cellulose
(Time Necessary to Complete Fermentation)
Concentration of
Sodium Sulfite
*
Culture 1
Culture 2
Culture 5
Days
Days
Davs
0.5
10 *
10 *
10 *
1.0
10 **
10 **
10 **
1.5
10 **
10 **
10 **
2.0
10
10 **
10 **
4- - 5
4- - 5
^ - 5
Control
(No 3ulfite)
Slight growth;
No growth
no decomposition
103.
at a temperature such as 60°C.
lfeuberg and Cohn (1 3 )
encountered analogous difficulties in their experiments
with sulfite at high temperatures.
The effect of sulfite when added after 3 days
of normal fermentation did not show any marked change
of fermentation.
Only O .5 per cent sulfite allowed
growth.
it appeared that once equilibrium
However,
was established between sulfite and the reacting
system,
the process was arrested.
In a study to determine whether acetaldehyde
could be fixed as an intermediate 2 liters of medium
were inocula,ted and incubated at 60°C for 3 days.
At
the end of this period enough sterile sodium sulfite
solution was added to bring its concentration to
0.5 per cent and incubated for 7 days.
The determin­
ation of fixed acetaldehyde was followed according to
the procedure of ileuberg and Reinfurth (5 3 ).
An
aliquot of liquor was treated with basic lead acetate
and excess calcium carbonate,
then distilled into
cold strong bisulfite solution.
The acetaldehyde
was determined by titra/tion of the distillate with
iodine by the Clausen method (4-9).
A qualitative
test for acetaldehyde was made
by the method of Klein and Pirschle
(511-)*
To 10 m l -
solution of the previously titrated aldehyde— sulfite
10M-.
solution,
3 ml*
solution and 2 ml,
^ P © 3? cent sodium nitroprusside
(l - 1 0 ) piperidine were added.
This reaction gives a blue-violet color if acetalde­
hyde is present in great quantities and a green color
if present in small concentrations
(l - 5000 to 1 - 1 0 ,0 0 0 ).
If the dilution is greater than 1 - 1 0 ,0 0 0 , no acetal­
dehyde is present under the conditions of the experiment
and the original yellow color is intensified.
Control
solution containing the same concentration of acetalde­
hyde as those found with titration were similarly
tested as was acetone.
In another series of experiments acetaldehyde
was distilled from the sulfite— fermentation liquor
into 2 , 4— dinitro-phenylhydrazine and dimedon solutions.
Recovery of the derivatives was attempted.
The distillate of each fermentation when
trapped in 2 , H-dinitrophenylhydrazine and dimedon
solutions gave slight turbidities.
Recovery of the
aldehyde derivatives was attempted but no satisfactory
results were obtained.
The yields of acetaldehyde produced per liter
of liquor are given in table XX.
determinations were made,
variable.
Although only a few
the amounts in each case were
Culture 2 was highest and culture 1 lowest.
105
Table XX
Yields of Aldehyde Determined by Titration
of Sulfite Complex with Iodine
No. of
Replicates
Culture
Acetaldehyde per Liter
Msyns.
1
2
O.lU- - 0.23
2
1
0.44
3
1
O .33
Table XXI
Qualitative Test of Acetaldehyde with
Sodi um Nitroprusside and Piperidine
Color
Culture
1
Control
2
Control
3
Yellowish sreen to lischt sreen
Dark ereen to bluish green
Shade darker than lip:ht screen
Bluish green
Lio-ht screen
Control
Bluish green
Acetone
Yellow
io6.
These values are not considered conclusive for it is
possible that acetone if present would give the same
reaction w ith sulfite.
However,
according to previous
analysis the yields of acetone were much higher.
Consequently,
if this is true in the above case, the
values of acetaldehyde should be much higher.
The color reactions indicated in table XXI
are not as sharp as the controls with the same aldehyde
concentrations.
However,
acetone with varying concen­
trations gave a distinct yellow color in each test.
The fact that slight turbidities were obtained
with 2 , H— dinitrophenylhydrasine and dimedon solutions
is indicative of a carbonyl compound.
It is recognized
that the latter reagent will react only with an alde­
hyde compound.
Neuberg and Cohn (1 3 ) obtained yields
as high as 0.0514- gm. of acetaldehyde by use of dimedon.
However,
this value was only obtained after 2> weeks of
incubation of the sulfite liquor at 56°C.
Although
acetaldehyde has not been definitely identified, all
the evidence tends to show that it may be an intermediary
in the mechanism of cellulose' breakdown.
107
.
I- Attempts to teat for the Presence of Cellulase
Enzvme:
1- Bactexia-fiee Filtrates:
Two liters of sterile medium were inoculated
and incubated at 60°G for 6 days.
Samples of liquor
were removed daily until the end of fermentation,
cooled,
and subjected to the following treatments.
(a)- Portions were filtered through Berkfeld,
Seitz, and Chamberlain filters.
(b)- Samples were concentrated under vacuum
at 37° "k0 ^ 5°G a-nc*. filtered as above.
(c)- Samples were treated separately w ith the
following reagents to purify the liquor by fractional
precipitation of proteins;
saturated ammonium sulfate
solution,
ammonium sulfate plus alcohol,
acetone, and
alcohol.
The precipitates were obtained by centrifuging
and taken up in small amounts of distilled water,
dialyzed against distilled water for 2k- to
at ice box temperature.
then
hours
The filtrates were dialyzed
against distilled water.
Liquor concentrated under vacuum at 37° "to 4-5°C
was similarly treated.
simultaneously run.
Controls without dialyzing were
log.
(d)- Portions of unfiltered and filtered
(as under
"a") liquors were treated with, activated
carbon at a pH range from 5.3 to 7.2 and placed in
the ice box over night.
The liquors were filtered
through filter paper and the residues treated with
phosphate solutions at the same p H range to determine
if elution of an active fraction was possible.
Part
of the filtrates and phosphate solutions were dialyzed
against distilled water at ice box temperature for
hours.
Similar treatment of liquor with kaolin
was performed.
(e) — Liquors
from fermentations with O . 5 ,
1 .0 , 2 .0 , and ^l-.O fo toluene were also treated as
stated in above procedures.
(f)— Test for the Enzyme:
To small test tubes containing sterile
phosphate buffer solutions* and tared filter paper
were added 1 to 3 ml.
Three ml.
of the enzyme preparations.
of the enzyme preparation were added to
M
1
* yjr phosphate solutions of pHs 5
7*^ were diluted
w ith distilled water at ratios of 2 to 1,
1 to 1,
and 1 to 2.
Two ml. of each solution were added
to small test tubes containing tared filter paper
and sterilized for 20 minutes at 15 pounds pressure.
109
test tubeB containing only tared filter paper.
cubation was at 3 7 , IJ-5 , and 60°C for
and anaerobically*
.
In­
days, aerobically
Reducing sugars were determined
before and after incubation by the method of Stiles,
Peterson,
and Fred (^3 ).
The filter paper was placed
in a tared Gooch crucible, washed with 5 per cent
hydrochloric acid, followed with distilled water and
dried at 1 0 5 °C for 6 hours and then weighed.
The enzymic hydrolysis of cellulose is
considered to be a degradation process by which
reducing sugars are produced with a corresponding
decrease of the substrate.
therefore,
In these experiments,
the hydrolysis was determined by the
measurement of reducing sugars and the residual
cellulose.
In all procedures without toluene,
there
was no evidence of enzymic hydrolysis of cellulose.
Reducing sugars and cellulose contents were not sig­
nificantly altered from the original.
The influence of toluene upon the enzyme
system of fermentation was only appreciably noticeable
with liquors when concentrated under vacuum.
Although
slight increases of reducing sugars were observed with
0 .5 , 1 , and 2 per cent toluene applications,
a k- per
cent treatment was found to be most effective.
110.
Purification of the liquors by adsorption or fractional
precipitation did not show any marked difference in
reducing sugars.
In like manner, dilute liquors
produced negligible results when compared with those
of concentrated liquors.
In general,
anaerobic
conditions proved to be superior to those of aerobic.
Dialysis did not appear to affect the activity of any
of the preparations.
Incubation at 60°C gave slightly
better results than at 37° or ^5°^.
Though very little
difference was noted between Seitz, Berkfeld,
Chamberlain filters,
and
the first was almost entirely
used throughout this work.
According to table XXII,
it is noted that
an increase of reducing sugars occurred within the
optimum pH range of cellulose breakdown,
to 6.0.
i. e . , 5*0
Though an increase of reducing substances
was observed, there was not enough decrease obtained
in the cellulose content to warrant any conclusion of
hydrolysis.
It is quite possible that small amounts
of cellobiose were present in the liquor at the begin­
ning of each determination and by the action of
cellobiase an increase of reducing sugars was obtained.
Since the enzyme, cellobiase,
functions best between
pH 5.0 and 6.0 and since no relative change of cellulose
111.
Table XXII
Enzyme Activity of Concentrated Bacterial
filtrates* from fermentations treated
with, bjo toluene
(incubation at 60°C for 6 days)
Under Anaerobic Conditions
Culture
pH
2
3
Final
Reducing
Sugars
per ml.
of liquor
means.
Ini tial
Cellulose
£ms.
Final
Cellulose
sms •
1.54-4-
1.636
0.0531
0.0538
3*3
1.4-98
1.531
0.0592
0.0386
5.6
1.336
1.963.
0.0567
0.0561
6.0
1.524-
1.667
0.0607
0.0609
6.2
1.333
1.568
0.054-6
0.054-8
3.0
1.266
1.302
0.0631
0.0626
3.3
1.24-7
1.-599
0 .06i4-
0.0611
3.6
1.263
1.731
0.0576
0.0581
6.0
1.271
1 .54-1
0.04-97
0.04-94-
6.2
1.226
1.84-9
0.0563
0.0568
3.0
0.64-2
0.890
0.04-76
0.04-88
3.3
0.623
1.236
0.0507
0.0516
3.6
0.636
1.14-1
0.0571
0.0572
6.0
0.601
0.807
0.0537
0.0539
6.2
0.636
0.674-
0.0658
0.0657
3.0
1
Ini tial
Reducing
Sugars
per ml.
of liquor
means.
..
* Liquor is from a five day fermentation period.
Concentrated under vacuum at *4-5°
thru a Seitz filter.
and filtered
112.
content was observed,
it is not probable that any
splitting of- cellulose took place.
Although several
workers have reported some evidence of cellulase
activity from bacterial sources,
their experimental
results are similar to those presented above.
2- Wet Bacterial Cells!
Bacteria,! cells from 16 liters of fermentation
liquor were obtained by use of a Sharpies super­
centrifuge.
The cells were frozen and thawed 6 times
and then triturated with sand.
One ml. of distilled
water was added to this mixture s n d mixed well.
Equal
j
portions were added to a series of test tubes containing
glycerol,
ethyl acetate,
CEirbon tetrachloride,
chloroform,
thymol, toluene,
5 $ sodium chloride solution,
and
distilled water,
r'espectively.
Each autolytic reagent
was tried at pH.
5*5>
7 >
6 .0 ,
and 7 *2 , and then
let stand 5 days at 3 7 °c > room, and ice b o x temperature.
At the end of this period each preparation was filtered
through a Chamberlain candle and the filtrate was tested
for its enzyme activity as previously given.
Filtration
with Seitz filters was also attempted.
In this study,
toluene was found to be the most
effective autolytic agent in producing highest yields
of reducing sugars.
Although thymol and chloroform
113.
gave slight: increases of reducing substances,
their
effect was negligible as compared to that of toluene
shown in table XXIII,
It was noted that a pH
6 .S - 7.2 for 5 days at 370 was best suited for
autolysis.
filtrates,
As with the experiments using bacterial
the rise of reducing sugars occurred within
the realm of optimum pH conditions for cellulose
breakdown.
In similar agreement,
there was no account­
able reduction of cellulose content obtained in any
of the experiments.
assumption,
Again these results support the
as indicated on page 1 1 0 , that the
enzyme which causes an increase of reducing sugars
may be due to cellobiase.
The fact that no marked
change of the substrate was observed and that the
increase of reducing substances was within the optimum
pH range of hydrolysis of cellobiose indicate the
action of the cellobiase enzyme rather than cellulase.
114-.
Table XXIII
Autolysis of Bacterial Cells by Toluene
(incubation at b0°C for 6 days)
under anaerobic conditions
Culture
1
2
3
.
Initial
Reducing
Sugars
means.
F inal
Reducing
Sugars
means.
Initial
Gellulose
eons.
Final
Cellulose
ems.
5.1
0.066
0.125
0.0510
0.0506
5.6
0.105
0.191
0.04-96
0.0502
6.0
0.095
0.109
0.0561
0.0551
5.1
0.125
0.14-2
0.0597
0.0561
5.6
0.14-2
0.201
0.0516
0.054-9
6.0
0.115
0.157
0.04-67
0.04-71
5.1
0.095
0.111
0.0516
0.0521
5.6
0.115
0.210
0.0566
0.0564-
6*0
0.105
0.14-2
0.0551
0.054-6
J&H
_
115
.
3“ Dried Bacterial Cells:
Bacterial cells from ^-0 liters of liquor were
dried under vacuum in a dessicator.
After 10 days the
cells were subjected to the same treatment as mentioned
above*
Tests were made for the presence of the enzyme*
All tests with previously dried organisms
were negative.
The amount of reducing substance was
smaller than that of wet cells*
The contents at the
end of the tests did not vary from the original.
Since no sign of enzyme action was obtained,
this
inactivity may be due to the conditions under which
the enzymes are destroyed.
Apparently the effect of
dessication does not favor the retention of active
bacterial substances*
5- Residue from Fermentation of Cellulose:
The residue from l6 liters of fermented
liquor was ground in a ball mill for six hours,
frozen
over night and ground for six additional hours.
This
was followed with 6 intermittent freezing and thawing
procedures.
The material was treated similarly to
the bacterial cells and the enzyme preparations were
likewise tested.
Table XXIV
Residue from Fermentation of Cellulose
Effect of toluene
(Incubation at 60°C for 6 days)
under anaerobic conditions
Culture
2
3
Final
Reducing
Sugars
mems •
Initial
Cellulose
CT1S.
Final
Cellulose
eras.
6.5
0.265
0.506
0.0*4-61
0.0*1-65
3.6
0.270
0.*4-*l-6
0.061*4-
0.0515 _
6.0
0.29*4-
0.721
0.0*4-66
0.0*4-65
5_.5
0.175
0.229
0.0672
0.0570
5.6
0.191
0.552
0.06*4-5
0.05*4-6
6.0
0.160
0.210
0.619
0.0517
6.5
0.229
0. 29*4-
0.0*1-56
0. 0*4-5*+
s .6
r3r
C\J
•
O
1
oH
Initial
Reduc ing
Sugars
rim'ms.
0.1+56
0.0556
0.065*4:
6.0
0.265
0.290
0.0669
0.0562
<
117.
As in previous experiments,
toluene was
found to be superior to the other autolyzing agents*
The conditions under which active preparations were
obtained were approximately similar*
As indicated
in table XXIV, practically analogous results were
obtained to those of bacterial cells and filtrates.
Consequently,
these tests likewise did not substantiate
the presence of cellulase enzyme.
In view of the above evidence, the following
interpretation is suggested in explaining the source
of the cellulase enzyme and its relation to hydrolysis
of cellulose.
First, the employment of various methods
produced no positive sign of cellulose breakdown
without living cells.
Second, an increa.se of reducing sugars
without a corresponding change in the substrate may
be attributed to the action of cellobiase rather than
cellulase enzyme.
Liebowitz
The fact that Pringsheim and
(3^) and Simola (3 3 ) reported cellulase
activity may be explained by the fact that their sole
criterion of hydrolysis was based on the increase of
reducing sugars.
Third, because the bacterial cell is apparently
concentrated on the surface of the cellulose present,
llg.
it is likely that the enzyme may be directly associated
with the bacterial cell rather than with bacteria—
free filtrate.
It is assumed that either the bacterial
cell possesses active enzyme centers on its surface
by which in intimate contact with cellulose hydrolysis
is initiated or through an intricate enzyme of the
cell functions in the process.
It is probable that
the latter process is unlikely since the complex
cellulose molecule could hardly diffuse through the
cell membrane.
SUMMARY
AND
CONCLUSIONS
SUMMARY AND CONCLUSIONS
The investigation on the thermophilic fermen­
tation of cellulose was divided into several phases.
Although a part of the work was limited to isolation,
cultivation,
and morphological studies, a greater
part was devoted to the conditions which most favored
cellulose breakdown.
It was also desired to investigate
the intermediary mechanism of the fermentation process
by use of poisoning and fixing agents.
Finally, attempts
were made to discover the possible source of cellulase
enzyme and its relation to cellulose hydrolysis.
1—
Three different types of thermophilic
fermentation of cellulose
(60°C) were observed when
crude cultures from horse manure were subjected to
successive transfers.
The first was characterized
by production of hydrogen sulfide and butyric acid,
the second by a yellow pigment, and the third by absence
of pigment or marked odor.
Maximum decomposition of
cellulose was obtained at 60°C after *4- to 5 days of
fermentation.
The enrichment cultures did not lose
any of their characteristics nor their capacity to
attack cellulose after two and one half years of culti­
vation w ith over 300 transfers in liquid medium.
2—
Growth of the cultures was obtained on
solid media under aerobic and anaerobic conditions
at 60° and slower development at 37°^.
Reversal of
oxygen and temperature conditions did not materially
affect the growth.
In each case,
the colonies on
plates were pin-point in size and transparent.
Growth on slants was filiform, somewhat beaded and
transparent.
Cellulose was attached by plate—picked
colonies and by those from deep tubes containing one
per cent agar.
Addition of glucose or cellobiose
solutions to the fermentation tubes stimulated the
activity of the process with the former being more
effective•
3- After 25 successive one day transfers,
the cultures attacked cellulose with slight loss of
properties.
medium,
However,
on subsequent transfers to new
the original characteristics of each culture
were restored.
4- Heat treatment of young and old cultures
at 105°C for 6 hours did not alter any characteristic
property of fermentation.
5- Storage of the cultures in the fermented
liquor for long periods at ice box temperature did not
121.
affect their capacity to attack cellulose.
6- The medium of Viljoen,
Fred, and Peterson
(Section B, medium B— l) was found best for producing
consistent results.
peptone
Organic nitrogen in the form of
(Difco) was necessary in promoting growth and
vigorous fermentation of cellulose.
Although Witte's
or Merck's peptone supported slight growth, no decom­
position of cellulose took place.
Cysteine was not
as effective as peptone for only slight decomposition
of cellulose was obtained.
alone support growth.
In no case did asparagine
It was found that inorganic
nitrogen and sulfur were necessary for best fermenta­
tion and that ammonium nitrogen was superior to nitrate
nitrogen.
7— The stimulating effect of salts on the
rate of fermentation was most pronounced with ferric
chloride and stannous chloride.
ous sulfate,
Zinc chloride, mangan-
ferrous sulfate, and sodium chloride were
less effective.
Lanthanum chloride, lithium chloride,
barium chloride,
sodium perborate,
and aluminum
chloride exerted a slight retarding effect on the
rate of fermentation.
6— The presence or absence of atmospheric
oxygen during fermentation of cellulose did not produce
a significant difference in the rate of reaction*
Cellulose appeared to be fermented equally well under
both conditions and there was no change in property
of the cultures*
Consequently,
the cultures may be
classified as facultative anaerobes.
9—
When the initial reaction of the fermentat
process was slightly above the neutral point,
the first
sign of cellulose breakdown was observed at approximately
pH 6.0;
sluggish.
below pH 6.75 fermentation was incomplete and
The optimum pH of the cultures for maximum
decomposition of cellulose was variable;
range was between pH 5.3 and 6.0.
however, the
Ag the initial re­
action was brought to the range of optimum cellulose
breakdown less of the substrate was fermented.
A
medium w ith an initial p H of 5.5 supported growth but
gave no cellulose hydrolysis.
Because best growth was obtained above pH
6.75 and subsequent breakdown of cellulose occurred
below this va.lue, it is assumed that the fermentation
process may take place in two stages.
The first consists
of rapid proliferation of the flora and the elaboration
123.
of substances necessary for cellulose hydrolysis and
the second the actual breakdown of cellulose at a
lower pH.
10- Young cultures (l — 7 days) completed
fermentation in 4- to 5 days and older cultures
IS months)
in A to 7 days.
However,
(6 -
the character­
istic properties of the older cultures were more
pronounced than those of the younger ones,
11- Young cultures (l - 6 days) when previously
shocked at 105°C for 15 minutes did not increase the
rate of fermentation.
Old cultures
(l - 6 months) were
markedly stimulated by heat treatment and completed
fermentation in 3 to ^ days.
12- The progressive morphological changes of
the cultures during fermentation of cellulose consisted
of the following sequences 1
(a)- Gram positive rods.
(b)— Vegetative cells, Gram reaction variable.
Appearance of granular cells that exhibited bipolar
staining.
A few terminal spores were present.
(c)- An increased number of terminal spores and
granular cells plus a few free spores.
negative cells.
A few Gram
12^.
(d)- An increased number of free and terminal
spores and relatively few granular cells.
(e) — Free spores resembling coccoid— like forms;
very few granular cells.
13- The cultures were able to ferment most
carbohydrates listed with acid but no gas.
lactose,
Galactose,
and raffinose gave an alkaline reaction.
14- _ Woody substances such as pine wood or
excelsior were very resistant to decomposition*
Towel
paper was only slightly decomposed and the fermentation,
in each case, was sluggish and incomplete.
Cotton was
readily fermented, but not as rapidly as filter paper.
No decomposition was observed on cacao shells.
15- The end-products of the thermophilic
fermentation of cellulose are acetic acid, butyric
acid, ethyl alcohol,
sugar (as glucose),
sulfide, hydrogen,
acid.
acetone, lactic acid, reducing
carbon dioxide, methane, hydrogen
and residual acid - i. e. succinic
Only culture 1 produced butyric acid and hydrogen
sulfide and no methane.
These products accounted for
gg - 92 per cent of the cellulose fermented under
nitrogen gas and 75 ~ 7^ per cent (carbon dioxide and
methane not included) under atmospheric conditions.
125.
Fermentation under nitrogen favored the breakdown of
cellulose by cultures 1 and 3 a-nd under atmospheric
conditions,
culture 2 was most effective.
Culture 1
was the most active cellulose fermenter, while culture 2
was more active than culture 3 under aerobic conditions
and less active under nitrogen.
Methane and carbon dioxide accounted for ap­
proximately 15 per cent of the cellulose fermented.
Maximum yields of gases were obtained during the second
day of fermentation.
The carbon dioxide - hydrogen
ratio during the progress of fermentation was variable
with each culture.
At the onset of fermentation the
'
ratio was low and at the end, it was high..
Glucose has been demonstrated as an end-product
of fermentation.
The sugar was identified by the pre­
paration of its osazone and, when calculated as glucose,
was equivalent to 37 to 4-2 per cent of the cellulose
fermented under anaerobic conditions and J>1 to 37 P er
cent under aerobic conditions.
l6-
The effect of toluene was characterized
by an accumulation of reducing sugars with an accompanying
reduction of neutral volatile compounds and volatile
acids as well as a relatively large decrease in the
amount of cellulose destroyed.
126 .
Breakdown of cellulose was obtained only when
the concentration of toluene was lowered to 0.5 per
cent.
Culture 2 was the most sensitive.
The changes
taking place in fermentation products after the ad­
dition of toluene suggest that this poison is most
effective at the stage of glucose breakdown.
17—
Fluoride was very effective in arresting
growth and hydrolysis of cellulose;
satisfactory
fermentation did not take place until the concentration
of sodium fluoride was lowered to 0.002 M.
1 and 2 were most sensitive to fluoride.
Cultures
No significant
abnormality was observed in fermentation products, but
rather a general depression in amount of growth and
cellulose fermented.
There is evidence that some
physiological adaptation of the organisms to ferment
cellulose at higher fluoride concentrations can take
place.
16-
The cultures were extremely sensitive to
sodium sulfite since a medium containing 0.5 per cent
of this compound supported growth but allowed no
decomposition of cellulose and with one per cent con­
centration,
growth was negative.
Evidence of the
presence of acetaldehyde as an intermediary of bacterial
glucolysis was found.
127.
19- Numerous attampts to obtain a cell-free
preparation of cellulase enzyme were unsuccessful.
It was possible to obtain preparations that would
increase sugar without changing the amount of cellulose.
Such a change was assumed to be due to the splitting
of cellobiose by the cellobiase enzyme.
20— All present evidence indicates an in­
separable connection of the cellulase enzyme with the
bacterial cell rather than a bacteria— free filtrate.
Since cellulose is an insoluble substance and since
the organisms are largely concentrated on the surface
of the fibers during fermentation,
it seems likely
that the initiation of hydrolysis takes place only
when actual contact is made between the cell and
cellulose particles.
BIBLIOGRAPHY
1- McBeth, I, G. and Scales, F. M.
U. S. Dept* Agr. Bur. Plant Ind* Bull., 266
(1913).
2- Kellerman, K. F. , McBeth, I. G. , Scales, F. ft . and
Smith, N. R.
Centbl. f. Bakt. Abt. II, 3 9 , 502
(1913).
3- Pringsheim, H. and Lichtenstein, S.
Centbl. f. Bakt. Abt. II, 60, 309
(1923-4).
4- Hutchinson, H. B. and Clayton, J.
Jour. Agric. Sci. 9, 14-5 (191S)*
5- G-ray, P. and Chalmers, C. H.
Ann. A p p l . Biol. 11, 324- (1924).
6- Lang we ll , H. and Hind, R.
J. Soc. Chem. Ind., 42, 327
(1 9 2 3 ).
7~ Langwell, H. end Lymn, A. H.
J. Soc. Chem. Ind., 42, 279
(1 9 2 3 ).
6-
Kroulik, A.
Centbl. f. Bakt. Abt.
II, 3 6 , 339
9- K h o u v d n ^ ,Y.
Ann. de I 1Inst. Pasteur, 37> ? H
Chem. Abs., 16, 261
(1924).
(1912-13)
(1 923 )*
10- Viljoen, J. A., Fred, E. B . , and Peterson, W . H.
J. Agr. Science, lo, 1
(1 9 2 6 ).
11- Pringsheim, H.
Zeitschr. f. Phys.
Chem.,
12- Pringsheim, H.
Centbl. f. Bakt. Abt.
13“ Neubepg,
Biochem.
II,
7^> 266
(1912).
3 6 ,513
(1913/*
C. and Cohn, R.
Z. 139» No* 7> 527 (1923)*
l4— Woodman, H. E.
J. Agr. Sci., 1 7 , 333
(1927).
15— Woodman, H. E. and Stewart, J.
J. Agr. Sci., IS, 713
(192S).
16— Woodman, H. E. and Stewart, J.
J. Agr. Sci., 22, 527
(1 9 3 2 ).
17— Coolhaas, S.
Centr. Bakt. Parasitenk.
IS-
Tetrault, P. A.
Centr. Bakt. Parasitenk.
II
Abt. 7 6 , 34-4-
II, SI, 2S
(192S)
(1 9 3 0 ).
19— Scott, S. W. , Fred, E. B. , and Peterson, W.. H.
Ind. Eng. Chem., 22, 731
(1 9 3 0 ).
20- Tomoda, Y.
J. Soc. Chem. Ind. Japan. Suppl. Vol. 35>
Chem. Abs., 2 7, IOS3
(1 9 3 2 ).
53^ B
(1932)
21 t- Tomoda, Y.
J. Soc. Chem. Ind. Japan. Suppl. Vol. 3&> ^3^ ®
Chem. Abs., 2 7 , 4-623 (1933).
(1933)
22- Langwell, H.
J. Soc. Chem. Ind.,
5 1 , 9^S
23- Snieszko, S.
Centbl. Bakt. Parasitenk.,
(1932).
II,
SS, 4-03
24- V e l d h u i s , M. K. , Christensen, L. M.
Ind. Eng. Chem., 2S, 4-30 (193^)*
25- Achorya,
Biochem.
2627-
G. N.
J. 24-, 14-59
(1933).
and Fulmer, E. I.
(1935)*
Peterson, W. H. and Snieszko, S.
Centr. Bakt. Parasitenk.
II, SS, 4-10
(1933)*
Waksman, 8 . H. and Hutchings, I. J.
Soil. Sci., 4-2, 119
(1 9 3 6 ;.
28- Olson, F. R. , Peterson, W. H . , and Sherrard, E. 1
Ind. Eng. Chem., 29» 1026 (1937).
29- Virtanen, A. I.
Rept. Proc. 3rd
Chem. Abs.
3^*»
Intern. Congr. Microbiol.
524-6 (194-0).
.^ r
1.1939 ).
130.
30— Simakora, T. L.
Arch.. Sci. Biol. (U.S.S.R.), 3 9 , 555
Chem. Abs.
30, 5609
(1936).
(1933).
31- Soetere, K.
Ann. Fermentations, 2 , 6 (1 9 3 6 ).
Annual Review of Biochemistry Vol. 6 , p. 6ll
32— Berl, E. and Koerber, W.
J. Amer. Chem. Soc., 6 0 , I5S5
(1937).
(193S).
33- Simola, P. E.
’'Uber den Abbau der Cellulose durch Mikroorganismen. 11
Thesis (2 vols.):
Helsinki
(l93l)»
Stephenson, M.
Bacterial Metabolism; 2nd Edition,
p. 67
(1939)•
3 *4—
35“
Pringsheim, H. and Liebowitz, I.
Zeitschr. f. Phys. Chem., 131, 262
(19 2 3 ).
Bradley, L. A. and Rettger, L. F.
J. Bact., 13, 321
(1927).
36- Cowles, P. B. and Rettger, L. F.
"J. Bact., 2 1 , 167
(l93l).
37“ C-rassmann, W. and Rubenhauer, H.
U. S. Patent.
Dec. 1*4-, 1937,
^^
0
^
French Patent.July 21, (193^)
39- Trager, J.
^
Biochem. J., 2 6 , 1 7 t>2
*4-0- Cray, P. H. H.
J. Bact.
1 2 , 273
2, 102, 315 .
7^7, 627.
(1932).
(1926).
*4-1- Matuszak, Maryan P.
Fisher Cas-Analysis Manual
Fisher Scientific Company,
(193^-)*
Pittsburgh, Penna.
*42- (a)- Popoff, L.
(b)- Honpe-Seyler, F.
(c)- Coolhaa.s, K. L.
(d)- S6hngen, N. L.
Bacterial Metabolism, pp. 132-133
1st Edition
by Stephenson, M.
(1930)
131.
Stiles, H .
J* Bact.,
,Peterson, W.
1 2 ,4-26
(1926).
, and Fred,
S.
B.
4-4-— Stalily, G. L. ,Osburn, 0. L. and Werkman,
The Analyst, 5 9 , 319
(1934-).
C.
H.
*4-3-
R.
H.
4-5- Werkman, 0. H.
Iowa State College Jr. of Sci.,
4-, 4-59
(1 9 3 0 ).
4-6— Goodman, L. F.
J. Am. Chem. Soc.,
4-2, 39
(1 9 2 0 ).
4-7— Osburn, 0. L. , Wood, H. G. , and Werkman, C. H.
Ind. Eng. Chem., Anal. Ed., 5 , 24-7 (1933).
4-6- Friedmann, T. E. and Graeser, J. B.
J. Biol. Chem., 1 0 0 , 291
(1933).
4-9- Clausen, S. W.
J. Biol. Chem.,
50— O'Meara.,
R.
5 2 , 263
(1 9 2 2 ).
A. Q.
J. Path. Bact., 34-, 4-01
(1 9 3 1 ).
51— Meyerhof, 0 . and Kiessling, W.
Biochem. Z.
2 6 3 , ^3
(1935)52— Meyerhof, 0. and Kiessling, W.
Biochem. Z.
261, 24-9 (1935)*
53— Neuberg, C. and Reinfurth, E.
Biochem. Z.
69, 365
(19IS).
54— Klein, G. and Pirschle, K.
Biochem. Z. , 1 6 6 ,
(1 9 2 6 ).
131
*4*3— Stiles, H.
J. Bact.,
4^1—
R. , Peterson, W. H. , and. Fred,
1 2 , 426
(1926).
E. B.
Stahly, G. L. , Osburn, 0. L. and Werkman,
The Analyst, 5 9 , 319
(1 9 3 4 ).
C. H.
45— Werkman, C. H.
Iowa State College Jr. of Sci.,
46— Goodman, L. F.
J. Am. Chem. Soc.,
42, 39
4, 459
(1930).
(1 9 2 0 ).
4-7- Osburn, 0. L. , Wood, H. G. , and Werkman, C. H.
Ind. Eng. Chem., Anal. Ed., 5 , 2^7
(1933)*
46— Friedmann, T. E. and Graeser, J. B.
J. Biol. Chem., 100, 2Q1
(1933).
49— Clausen,
J. Biol.
S. W.
Chem.,
50— O'Meara,
J. Path.
R. A. Q.
Bact., 34,
5 2 , 263
401
(1 9 2 2 ).
(1 9 3 1 ).
51— Meyerhof, 0. and Kiessling, W.
Biochem. Z.
263, S 3
(1935)*
52— Meyerhof, 0. and Kiessling, W.
Biochem. Z.
261, 249
(1935)*
53— Neuberg,
Biochem.
C. and Reinfurth, E.
Z.
6 9 , 3^5
(l9lB).
64— Klein, G. and Pirschle, K.
Biochem. Z. , l 6 S, ^k-0
(1 9 2 6 ).
Документ
Категория
Без категории
Просмотров
0
Размер файла
4 904 Кб
Теги
sdewsdweddes
1/--страниц
Пожаловаться на содержимое документа