the amino acid and monosaccharide content of the cell
TRANSCRIPT
Scholars' Mine Scholars' Mine
Doctoral Dissertations Student Theses and Dissertations
1967
The amino acid and monosaccharide content of the cell walls of The amino acid and monosaccharide content of the cell walls of
Thiobacillus thiooxidans Thiobacillus thiooxidans
Edward Hibbert Crum
Follow this and additional works at: https://scholarsmine.mst.edu/doctoral_dissertations
Part of the Chemical Engineering Commons
Department: Chemical and Biochemical Engineering Department: Chemical and Biochemical Engineering
Recommended Citation Recommended Citation Crum, Edward Hibbert, "The amino acid and monosaccharide content of the cell walls of Thiobacillus thiooxidans" (1967). Doctoral Dissertations. 1874. https://scholarsmine.mst.edu/doctoral_dissertations/1874
This thesis is brought to you by Scholars' Mine, a service of the Missouri S&T Library and Learning Resources. This work is protected by U. S. Copyright Law. Unauthorized use including reproduction for redistribution requires the permission of the copyright holder. For more information, please contact [email protected].
THE AMINO ACID AND MONOSACCHARIDE CONTENT OF THE CELL
WALLS OF THIOBACILLUS THIOOXIDANS
by
EDWARD HIBBERT CRUM -· / <i ... · '
A DISSERTATION
Presented to the Faculty of the Graduate School of the
UNIVERSITY OF MISSOURI AT ROLLA
In Partial Fulfillment of the Requirements for the Degree
DOCTOR OF PHILOSOPHY
1967
. I
/' '
~-~~~---: I
THE AMINO ACID AND MONOSACCHARIDE CONTENT
OF THE CELL WALLS OF THIOBACILLUS THIOOXIDANS
By: Edward Hibbert Crum Advisor: Dr. Donald J. Siehr
ABSTRACT
The objectives of this investigation were: (a) to
ii
culture large quantities of Thiobacillus thiooxidans cells,
(b) to find an acceptable method of rupturing the cells,
(c) to find an acceptable method of isolating the cell walls,
and (d) to analyze the cell wall components, especially the
amino acid and sugar components. The strain of T. thiooxidans
used for this investigation was American Type Culture Col-
lection Number 8085.
The surface active agents Tergitol 08* and Tween 80**
were investigated for their ability to reduce the lag period
found in the growth of T. thiooxidans. Although a concentra-
tion of 12.5 ppm Tween 80 did decrease the lag period, the
use of these surfactants was not effective in increasing the
amount of cells grown over a specific time period because
they reduced the cell yield.
Two cell rupture techniques, sonication and pressure
cell rupture, coupled with either washing with deionized
Edward H. Crum
*Union Carbide Corp. Registered Trademark.
**Atlas Chemical Industry Registered Trademark.
iii
water or washing and treatment with IR-120* and AG-1X2* ion
exchange resins, were tested. The cell walls were also
ruptured in both deionized water and deionized water
buffered at pH 7.9. The release of soluble nitrogen was
used as a measure of the effectiveness of cell rupture. The
best rupture of T. thiooxidans was obtained by washing the
cells with deionized water, followed by pressure cell rupture
in the basic buffer solution. The procedure solubilized
68% of the total nitr~gen present.
The cell walls were isolated and purified by a combina-
tion of differential centrifugation, linear sucrose gradient
centrifugation, and washing. The cell walls were hydrolyzed
in sealed tubes at 100 to 120 C with hydrochloric acid,
sulfuric acid, and the ion exchange resin IR-120 as catalysts.
In the study of the amino acids in cell walls, the walls
were also digested with trypsin, washed, and then hydrolyzed
with hydrochloric acid in a sealed tube. The cell wall
hydrolyzates were separated by two dimensional paper
chromatography for a qualitative estimation of the amino
acids present in the cell walls. A quantitative determina-
tion of the amino acids in the cell wall hydrolyzates was
obtained by the use of an Amino Acid Analyzer.
The cell wall of T. thiooxidans was found to have an
amino acid content qualitatively and quantitatively more
*Obtained from Bio-Rad., 32nd and Griffin Streets, Richmond, Calif.
Edward H. Crum
like that of Gr~m-negative than Gram-positive bacteria.
Evidence that ornithine was present in the cell walls was
obtained. The ornithine might possibly have been an
artifact formed during the rupture procedure.
Hydrolysis of the cell walls in sulfuric acid failed
to liberate monosaccharides. Therefore, hydrolysis of the
cell walls in the presence of IR-120 was used for qualita
tive determination of the monosaccharides present in the
iv
cell wall. Paper chromatography of the cell wall hydrolyzates
indicated the presence of rhamnose, glucose, and galactose.
The hydrolyzates also contained small amounts of the amino
sugar glucosamine. Since the resolution of glucose and
galactose was not good on papergrams, the monosaccharide
mixture was treated with glucose oxidase before chromatography.
The gluconic acid formed with this treatment was nicely
separated from the galactose.
Since the cell walls of T. thiooxidans enables the
bacterium to survive in very acidic media, knowledge of the
cell wall composition has implications in the control of acid
mine drainage by providing a rational approach to the pre
vention of the growth of this organism.
Edward H. Crum
TITLE PAGE
ABSTRACT . .
TABLE OF CONTENTS
. . . . . . . . . . . . . . . . . . . . . . . . . .
. . .
TABLE OF CONTENTS
LIST OF FIGURES
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
LIST OF TABLES . . • . . . . . . . . . . I. INTRODUCTION ... . . . . . . .
II. LITERATURE REVIEW .. . . . . . .
III.
A.
B.
THIOBACILLUS THIOOXIDANS
THE BACTERIAL CELL WALL .
. . . . . .
EXPERIMENTAL . . . . . . . . . • . . .
A. MATERIALS AND METHODS . . . . • . . .
1. Cultivation of Cells ...•...
2. Preparation of Cell Walls .
. . .
. . .
. . .
v
Page
~
ii
v
vii
ix
1
6
6
18
41
41
41
44
3. Cell Wall Components. . . . . . . . . . 50
B.
4. Protein and Nitrogen Determinations 54
5. Surfactant Studies . . . . . . . . . . 57
RESULTS . . • . . . . . . . .
1. Surfactant Studies .•..
2. Cell Rupture .•....••...
3. Paper Chromatography ...... .
. . . 59
59
64
64
4. Amino Acid Analysis . . . • . . . . . . 6 8
5. Test for Contamination •.. . . . . 68
vi
Page
IV. DISCUSSION . . . . . . . . . . . . . . . . . . . 72
A. DISCUSSION OF RESULTS . . . . . . . . . . . 72
1. Surfactant Studies . . . . . . . 72
2. Cell Rupture . . . . . . . . . . . . . . 74
3. Hydrolysis Methods . . . . . . . . . . . 76
4. Analysis of Cell Walls . . . . . . . . . 77
5. Test for Contamination . . . . . . . . . 87
B. LIMITATIONS. . . . . . . . . . . . . . . . . 88
v. CONCLUSIONS. . . . . . . . . . . . . . . 90
VI. RECOMMENDATIONS . . . . . . . . . . . . . . . . 91
VII. BIBLIOGRAPHY . . . . . . . . . . . . . . 92
VIII. ACKNOWLEDGEMENT. . . . . . . . . . . . . . . 101
IX. VITA . . . . . . . . . . . . . . . . . . . . 102
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
vii
LIST OF FIGURES
Page
Proposed Scheme of Carbon Dioxide Fixation by Thiobacillus thiooxidans (56) . . . 14
Chemical Composition of Bacterial Cell Walls. . . . . . . . . . . . . . . 20
Diagrammatic Representation of Cell Walls (29, p. 23) . . . . . • . . . . . . . . . 21
Structures of a- E Diaminopimelic Acid and Muramic Acid (76, 77, 78)....... 24
The Proposed Structure of Amino Sugar, Di-and-Tetrasaccharides from Micrococcus lysodeikticus (82) . . • . . • . . . . . . 26
Type of Molecular Structure Proposed for the Cell Wall of Micrococcus l;'isodeikticus ( 8 3) . . . . . . . . . . . . . . . 27
Structure of a "Park Nucleotide" from StaEh~lococcus aureus (25) . . . . . . 29
Figure 8. Structure of Two Glycosaminopeptides Isolated from Escherichia coli Cell Walls (88) . . . . . . . . . . . . . . . . . 31
Figure 9. Hypothetical Glycosaminopeptide Subunit of the Cell Wall of Micrococcus lysodeikticus (86) • • • . • • • • . . • • . . • 32
Figure 10. Hypothetical Subunit of the Cell Wall of Aerobacter cloacae (29, 147) . . . . • . . 33
Figure 11.
Figure 12.
Figure 13.
Figure 14.
Four General Types of Teichoic Acid (29' p. 159) . • . . . • . . ..
Proposed Structure of Cell Walls of Staphylococcus aureus (89) ..•
General Scheme for Differential Centrifugation (29, po 58) . o •• o o •••
Sketch of Fermentation Apparatus (91)
Figure 15. Device Used to Produce Linear Sucrose Gradients o . o • • o o o • • • • • •
34
35
0 • 39
0 0 43
47
Figure 16. Layers Formed During Linear Sucrose Gradient Purification . • . • • • . . . .
Figure 17. The Effect of Surfactant Tween 80 on the Growth of Thiobacillus thiooxidans.
viii
Page
49
Shake Flask Studies • . • . • . . • • • • 61
Figure 18. The Effect of Surfactant Tween 80 on the Growth of Thiobacillus thiooxidans. Shake Flask Studies • . . . • . . . . • . 62
Figure 19. The Effect of Surfactant Tween 80 on the Growth of Thiobacillus thiooxidans• Fermentor Studies . . • . . • . • • • . . 65
Figure 20. Ultraviolet Absorption of Cell Walls and Supernatant from Pressure Cell Rupture. . 71
ix
LIST OF TABLES
Page
Table 1. Principal Classes of Chemicals Found in Cell Walls of Bacteria (29, p. 251) . . 23
Table 2. The Effect of Surfactant Tween 80 on the Growth of Thiobacillus thiooxidans. Shake Flask Stud~es . . . . . . . . . . • . 60
Table 3. The Effect of Surfactant Tween 80 on the Growth of Thiobacillus thiooxidans. Fermentor Stud~es . . . . . . . . . . • • . 63
Table 4. Thiobacillus thiooxidans Cell Rupture Experiments . . . • . . . . . . . . . . . .
Table 5.
Table 6.
The Qualitative Amino Acid Content of Thiobacillus thiooxidans Cell Walls . .
The Quantitative Amino Acid Content of Thiobacillus thiooxidans Cell Walls
Table 7. The Qualitative Amino Acid Content of Some Gram-positive and Gram-negative
. .
. .
66
69
70
Bacterial Cell Walls ( 9 5) • . • . . . . 79
Table 8. The Quantitative Amino Acid Content of Some Gram-positive and Gram-negative Bacterial Cell Walls (29, p. 254-255} . . . 82
Table 9. Molar Ratios of Some Amino Acids in Cell Walls . . . . . . . . . . . . . . . . . . . 84
I. INTRODUCTION
With the advent of state and federal regulations regard
ing water pollution one serious problem of concern in the
Appalachian region is that of acid mine drainage. While
microorganisms may not be completely responsible for this
acid drainage, they act to speed the production of acid (1).
The organism Thiobacillus thiooxidans is generally found in
acid mine water. This fact is substantiated by the statement
of Temple and Colmer (2). "A survey of acid mine waters of
the Morgantown area, from other parts of the state [W. Va.]
and from Pennsylvania showed that Thiobacillus thiooxidans
is present 1n all the acid mine waters examined and may
reasonably be supposed to be in all acid mine waters." Many
other investigators have linked T. thiooxidans, as well as a
very similar organism Thiobacillus ferrooxidans, with acid
mine drainage or acid formation from pyrites in the soil (1,
3-17) .
In a survey made of the streams in West Virginia, it has
been estimated that an average of 168,349,000 gallons of mine
drainage containing the equivalent of 2,876,000 pounds of
concentrated sulfuric acid were daily flowing into the river
systems of the state (18). The total sulfuric acid equiva
lent for the Ohio River Basin was estimated at around
2,500,000 tons per year during the 1942 Ohio River Pollution
Survey (19).
The damage caused by acid mine drainage to navigational
and floating equipment in the Pittsburgh area was estimated
in 1926 by the U. S. War Department at 500,000 to 600,000
dollars per year (20). In 1942 the Works Progress
Administration made a conservative estimate of the extra
costs due to damage by acid mine drainage in the states of
the Ohio River Basin, and placed these costs at about
10,000,000 dollars per year (19).
2
Braley (1) recently reported that four methods are cur
rently used to treat or prevent acid mine drainage: (a) mine
sealing, (b) neutralization of the acid, (c) pickup system,
and (d) subsurface disposal of acid mine water.
The first method can be used with abandoned mines only,
while the last three are basically directed toward use in
operating mines, but could be used at abandoned mines.
According to Braley (1) , mine sealing has not been as
effective as originally expected because of "breathing''. He
explained that "breathing" occurs because of the natural
porosity of geological formations and fractures induced in
the strata above the coal.
Neutralization of the acid mine drainage was tried
during World War I by the Frick Coal Company. The project
was abandoned after the war as economically unfeasible (1).
The Sanitary Water Board of the Commonwealth of Pennsylvania
came to the same conclusion in 1951 (1). However, the
Commonwealth of Pennsylvania is now conducting an experiment
known as "Operation Yellow-boy" to study the feasibility of
neutralization of acid mine drainage. The most recent
report (21) states that it costs 1.09 dollars per thousand
3
gallons to treat acid mine drainage. This estimate was based
on data taken from a 240,000 gallon per day pilot plant, and
did not include additional expenditures for sludge processing.
Braley (1) has described a pickup system in which water
is picked up at the point of entry into the mine and trans
ported through pipes to the exterior of the mine. This
prevents the water from carrying away any acid formed. This
method has been reported as a success. Braley, however, did
not indicate the cost of such an operation.
Linden and Stefanko (22) have described some of the
problems which must be overcome in order to make subsurface
disposal possible. Some of these are: (a) the bacteria
present in the waste must be killed, (b) dissolved gases
must be removed, (c) suspended particles must be removed,
and (d) an exacting geological survey must be made before the
well is drilled. Linden and Stefanko have not experimentally
tried subsurface disposal, therefore cost data is not avail
able.
There are problems associated with each of the above
mentioned methods .. Sealing of a mine is applicable where
"breathing" is not a problem. Sealing would not, however,
solve the acid drainage problem of operating mines. Neutrali
zation is very expensive and causes another problem, disposal
of the sludge formed. The pickup system, while applicable to
operating mines, would not be effective in abandoned mines.
Subsurface disposal requires extensive treatment of the waste
before ultimate injection.
All the methods discussed above, except mine sealing,
are directed toward destroying or otherwise treating the
acid after it has been formed. The author feels that a
better approach would be to eliminate the production of
acid. Prevention of the growth of the microorganisms
responsible for the formation of the acid seems to be a
reasonable solution to the problem. This could be done by
spraying the mine surface with a substance which would
prevent the growth of the microorganisms.
4
The organism T. thiooxidans can live in solutions with
a very high acid concentration. The enzymes of T. thiooxi
dans, however, operate at nearly neutral conditions. This
indicates that the cell wall has some method for maintaining
the internal pH. T. thiooxidans can also utilize sulfur as
an energy source. Since sulfur is insoluble in water the
organism has some method for transporting the sulfur across
the cell wall. It is known that the action of penicillin is
directed toward the inhibition of bacterial cell wall
synthesis (23-28; 29, p. 15, 88, and 203-216). The
penicillin inhibits the formation of glycosaminopeptides.
A knowledge of the cell wall of T. thiooxidans may help in
selecting an inhibitor of cell wall synthesis or a chemical
which would modify the function of the cell wall and either
destroy its pH maintenance system or its sulfur transport
system.
The purpose of this work was: (a) to culture large
quantities of T. thiooxidans cells, (b) to find an acceptable
method of rupturing the cells, (c) to find an acceptable
method of isolating the cell walls, and (d) to analyze the
cell wall components, especially the amino acid and sugar
components.
5
6
II. LITERATURE REVIEW
This literature review is divided into two parts. The
first section relates background material concerning
Thiobacillus thiooxidans, the organism under study; the
second section deals with the bacterial cell wall and its
preparation for analysis.
A. THIOBACILLUS THIOOXIDANS
The organism under study, T. thiooxidans, has been
classified in Bergey's Manual of Determinative Bacteriology
(30) in the following manner:
CLASS
ORDER
FAMILY
TRIBE
~NW
SPECIES
SCHIZOMYCETES
EUBACTERIINEAE
NITROBACTERIACEAE
THIOBACILLEAE
THIOBACILLUS
THIOOXIDANS
Waksman and Joffe (31) were the first to isolate this
organism in pure culture. They reported their findings in
1920.
T. thiooxidans organisms are short rods, 1.0 ~long
and 0.5 ~in diameter, with rounded ends. They occur singly,
in pairs, and occasionally in chains. They are motile by
means of a singular polar flagellum (30, 32) ·
Waksman and Joffe (33) have reported that the organism
is Gram-positive, while starkey (34) has reported that it
is Gram-negative. Umbreit, vogel, and Vogler (35) explained
that this phenomenon was due to the conditions under which
the cells were stained; under acid conditions the organisms
reacted as Gram-positive, while under alkaline conditions
the organisms were found to be Gram-negative.
7
T. thiooxidans is autotrophic (30). By autotrophic, it
is meant that it has the ability to live and multiply in an
environment containing carbon dioxide as the sole source of
carbon; however, other sources of nutrients are required for
growth. Waksman and Joffe (33) gave the mineral require
ments for T. thiooxidans as mere traces of potassium,
magnesium, calcium, iron, and phosphorus. A nitrogen source
was also required for growth.
Carbon added in the form of carbonates was found to
inhibit growth, because it made the medium alkaline (33).
Bicarbonates in small amounts were found to support growth.
However, the presence of bicarbonates did not give a better
growth than that observed in samples incubated under the
same conditions without them (33). No organic compound has
been found which can substitute for carbon dioxide (36).
Starkey (37) has shown, however, that glucose may enter
into the metabolism of the cell in the presence of sulfur,
but glucose would not support the growth of the organisms
without the presence of sulfur. Starkey also found citric
acid in concentrations of 5% or higher inhibitive, while at
a 2.5% concentration growth was active. Butler and
Urnbreit (38) have shown the following compounds to be
absorbed by T. thiooxidans: acetate, glycerol, D-glucose,
glycine, DL-alanine, DL-aspartic acid, and DL-Proline.
Succinic acid, however, was not absorbed by T. thiooxidans.
Waksman and Joffe (33) found that glycerol, alcohol, man
nitol, and glucose acted as stimulants for growth. O'Kane
(39) has established the following growth factors to be
present in T. thiooxidans: nicotinic acid, pantothenic
acid, biotin, riboflavin, thiamin, and pyridoxine.
T. thiooxidans is strictly aerobic (30, 33, 37). It
obtains its nitrogen from ammonium salts. Nitrogen in the
form of nitrates has been found to be toxic. The nitrogen
of asparagine, urea, and peptone has been found to be
unavailable to this organism (30, 33, 37).
T. thiooxidans can utilize either sulfur or thiosulfate
as an energy source. Thiosulfate, however, retards growth
at concentrations between 3 and 10% (40). Waksman and
Joffe (33) reported that thiosulfate was capable of sup
porting growth, but to a much smaller extent than sulfur.
They also stated that hydrogen sulfide was not utilized as
an energy source by this organism. In opposition to this
view, Parker and Prisk (41), Waksman and Starkey (32),
Suzuki and Werkman (42), and Waksman (43) have reported that
hydrogen sulfide can be utilized by T. thiooxidans. Prisk
and Parker (41) also presented evidence which indicates
thiosulfate becomes toxic in concentrations above 1%.
Since sulfur and thiosulfate are the two energy sources
generally used in media, it is apropos that their oxidation
be discussed. There seems to be a general agreement that
8
the oxidation of sulfur takes place without the formation
of intermediates which accumulate in the medium, and that
sulfate is produced directly (15, 32, 33, 41, 44). The
reaction may be represented as follows:
Agreement as to the oxidation of thiosulfate is, however, a
different matter. Two reports indicated that no intermedi
ates were found (32, 43). The following equation was given
for the reaction:
Suzuki and Werkman (42) have reported polythionates as
intermediates in the oxidation of thiosulfate. Prisk and
Parker (41) have reported tetrathionate as an intermediate
and gave the following two-step reaction to explain their
findings:
6Na2s 2o3 + 50 2 4Na2so 4 + 2Na2s 4o6
In trying to explain these conflicting reports, Prisk and
Parker (41) suggested that the discrepancy may have come
about because of different sampling techniques. They found
that tetrathionate was only present during the 24 hour
9
period between the fifth and sixth day of incubation. They
noted that most of the other authors had taken their initial
samples after this period. Starkey(40) reported that below
a 0.5% concentration thiosulfate oxidation proceeded in a
quantitative manner to sulfates, but above this concentration
sulfur precipitated due to interaction of thiosulfate with
sulfuric acid present.
The optimum temperature for growth has been reported
to be in the range of 28 to 30 c. The organism grows
slowly at 18 and 37 C. A temperature of 55 to 60 c has
been found to kill these organisms (30, 33).
10
The optimum pH for the growth of T. thiooxidans has
been reported to be in the range of 2.0 to 3.5 (33, 37, 45),
while they can grow in the range 0.5 to 6.0 (30).
Carbon dioxide fixation, endogenous respiration, and
the ability to store energy in the absence of carbon dioxide
are areas in which there is quite a bit of disagreement.
Vogler (46) has reported an endogenous respiration quotient
a0 (N) (microliters of oxygen per milligram of bacterial 2
nitrogen) of 10 to 40. This indicates one of two possibili-
ties: (a) the cells had enough unoxidized substances stored
to require 10 to 40 microliters of oxygen to oxidize it, or
(b) some cells died and lysed; these lysed products were
then absorbed by other cells and oxidized.
Vogler (47) has reported that T. thiooxidans could
oxidize sulfur in the absence of carbon dioxide and store in
the cell, energy which could be used for carbon dioxide fixa
tion when sulfur oxidation was impossible. In the same
study it was observed that carbon dioxide was taken up under
anaerobic conditions by young cells. The young cells (7 day
incubation) were able to fix up to 40 microliters of carbon
dioxide per 100 micrograms of bacterial nitrogen without the
presence of sulfur. Older cells (14 day incubation) gave
off carbon dioxide in the absence of sulfur.
11
Vogler and Umbreit (48) reported that in the absence
of carbon dioxide, phosphate was transferred into the cells
when sulfur was oxidized. In the presence of carbon
dioxide and absence of sulfur the cells fixed carbon
dioxide and excreted phosphate back into the medium. The
authors suggested a link between sulfur oxidation and the
formation of phosphate esters, which in turn was linked
to carbon dioxide fixation.
Along the same lines, LePage (49) has reported that a
polysaccharide was formed and stored during sulfur oxida
tion. He suggested that the degradation of this polysac
charide was the source of carbon dioxide during endogenous
respiration. He also found a release of phosphate during
endogenous respiration. The chemical composition of this
polysaccharide was given as follows:
Carbon 43.25%
Hydrogen 7.86%
Oxygen 49.89%
Phosphorus 0.68 to 1.65%
Nitrogen 0.82%
LePage and Umbreit (50) have reported that fructose
-1, 6-diphosphate, phosphoglyceric acid, fructose-6-
phosphate, glucose-6-phosphate, and glucose-1-phosphate
were other phosphorylated compounds found in T. thiooxidans.
LePage and Umbreit (51) later reported the occurrence of
12
adenosine-3'-triphosphate in T. thiooxidans. This is an
unusual type of adenosine triphosphate and has not been
found in other organisms. Barker and Kornberg (52) found
no evidence of adenosine-3' or 2'-triphosphate in the cells
of T. thiooxidans.
Baalsrud and Baalsrud (53) using manometric techniques
have been unable to find carbon dioxide assimilation in the
absence of an energy source. They also reported results
which showed that phosphate was returned to the medium when
a cell suspension was not exposed to carbon dioxide. This
tends to invalidate the data presented by Vogler and
Umbreit (48).
On reexamination of previous experiments using radio-
active isotopes, Urnbreit (54) found, that sulfur oxidation
in the absence of carbon dioxide permitted cells to fix
carbon dioxide under anaerobic conditions. He also reported
the amount of carbon dioxide fixed was proportional to the
amount of sulfur previously oxidized. He reported the
release of phosphates when carbon dioxide was applied
anaerobically.
. 32 14 Newburgh (55), us1ng P and C 02 , reported results
which agreed in part with both the Baalsrud and Urnbreit
school of thought. He found carbon dioxide to be fixed by
the cells in the absence of sulfur, very little difference
in aerobic and anaerobic carbon dioxide fixations, and no
release of phosphate during carbon dioxide fixation. He
reported that p3 2 was taken up by the cells only when
13
carbon dioxide, oxygen, and sulfur were present simultane
ously.
Suzuki and Werkman (42, 45, 56-59) have made many
contributions to the understanding of T. thiooxidans
metabolism during their studies on carbon dioxide fixation.
They prepared cell-free extracts which in the presence of
glutathione oxidized sulfur. This was taken as evidence
for the presence of glutathione reductase in cell-free
extracts of T. thiooxidans. The glutathione reductase was
found to be similar to that present in Escherichia coli
( 60) •
In the presence of c14o2 , adenosine triphosphate and
ribose-5- phosphate cell-free extracts produced carboxyl
labeled phosphoglyceric acid (58). This reaction was
found to require the presence of magnesium ions and the
presence of phosphoglyceric acid was evidence for the
presence of phosphoribuloseisomerase, phosphoribulokinase,
and carboxylating enzymes in the T. thiooxidans metabolic
system.
Using c14o2 labeling, Suzuki and Werkman (56) found
the first product of carbon dioxide fixation to be phospho
glyceric acid. The enzyme phosphoenolpyruvate carboxylase
was also found to be present in T. thiooxidans by Suzuki
and Werkman (45, 57). From these findings these investiga
tors proposed a scheme, shown in Figure 1, for carbon
dioxide fixation by T. thiooxidans (56).
14
Hexose Phosphate
t *COOH *COOH *C02 Ribulose I
H-C-NH I 2
I --~---------~--H-c-oH Diphosphate
CH 20H
Serine
*COOH
I H-CNH 2
I CH2 I
b.COOH
Aspartic acid
I CH20P03H2
Phosphoglyceric acid ~
*COOH I "OP03H2
CH 2
Phosphoenolpyruvic acid t
*COOH I c=o I CH 3
Pyruvic acid
*yOOH
t=O I CH 2 I
b.COOH
Krebs Cycle
Oxalacetic acid
*CO 2
*CO 2
COOH I CH 2 I CH 2 I C=O I
b.COOH
Keto glutaric acid
COOH I IH2
CH 2
HC-NH 2
b.COOH
Glutamic acid
Figure 1. Proposed Scheme cf Carbon Dioxide Fixation by Thiobacillus thiooxidans (56).
15
There has been much speculation as to the method by
which insoluble sulfur is metabolized by the cell. Umbreit,
Vogel, and Vogler {35) have proposed that sulfur is taken
into the cell by solution in fat globules located at the
ends of the cell. This theory was opposed by Knaysi (61) on
cytological grounds.
Starkey, Jones, and Frederick (62) have postulated that
sulfur was enzymatically transformed into a transportable
form at the surface of the cell. In a study of agitation
and wetting agents, they found that growth was more rapid
in agitated flasks than in static flasks. They also
reported that the wetting agents Tergitol 08* and Tween 80**
in small concentrations increased the rate of sulfur oxida
tion.
Jones and Starkey (63) suggested that the amino acids
and polypeptides which they found in the medium wet the
sulfur and thus produced a more rapid oxidation of sulfur.
They made these observations while studying the surface
tension of the medium during the growth of T. thiooxidans.
They felt that the amino acids and polypeptides leaked out
of the actively metabolizing cells.
Vogler and Umbreit (64) have reported that T. thiooxidans
must be directly in contact with the sulfur for oxidation
to take place. In the same report the authors concluded
*Union Carbide Corp. Registered Trademark
**Atlas Chemical Industry Registered Trademark
16
that it was neither necessary nor probable that T. thiooxi
dans was able to render sulfur soluble in the medium. It
was found that surface active agents were capable of
increasing the rate of sulfur oxidation.
Schaeffer (65} has reported that 40 to 60% of the
material excreted by T. thiooxidans was a surface active
agent which he was able to extract from the medium. He
identified this material as phosphatidyl inositol. Jones
and Benson (66), however, have reported that the surfactant
was phosphatidyl glycerol and not phosphatidyl inositol.
They found no phosphatidyl inositol in either the total
cell extract or the lipid fractions. Although these results
do not agree as to the compound responsible, they do agree
on the point that a surfactant produced by T. thiooxidans
does aid in sulfur oxidation.
Reports indicate that increasing the partial pressure
of oxygen, above the medium, gave little increase in the
rate of sulfur oxidation (64, 67). Increasing the partial
pressure of carbon dioxide, however, increased the rate of
sulfur oxidation (40). Slight increases in the total
pressure also enhanced the oxidation of sulfur (40).
T. thiooxidans is of academic interest for several
reasons. The organisms are autotrophic. They must, there
fore, synthesize all required organic material from carbon
dioxide and inorganic salts. This requires metabolic steps
not found in heterotrophic organisms and therefore they may
be different from any heretofore studied. The organism can
17
by some means oxidize sulfur or certain sulfur containing
compounds to sulfuric acid and in this manner trap energy.
This energy is then used for cell growth and carbon dioxide
assimilation. This process is somewhat similar to photo
synthesis, in that the starting and final products are
known (sulfur and sulfuric acid), but the mechanism of
trapping the energy has not been elucidated. Another inter-
esting point is the fact that this organism can grow in an
environment of very low pH while maintaining its internal
pH near neutrality. Recently Fischer et al. (68, 69) have --observed that T. thiooxidans makes an electrical contribution
to a half-cell electromotive force when actively oxidizing
sulfur. At present the voltages are small but this research
may ultimately lead to a practical biocell.
18
B. THE BACTERIAL CELL WALL
The study of bacterial cell walls is not an old field.
Martin (25) indicated that for experimental purposes it
began in 1951 with a report by Salton and Horne (70) which
outlined a procedure for the isolation of cell walls on a
preparative scale.
Bacterial cell walls provide the vital bacterial
cytoplasm with a rigid, protective covering. Although the
covering provides rigidity to the bacterium, it allows for
the permeability of nutrients ranging from ions to mole
cules with molecular weights in the millions. The cell
wall is flexible enough to allow cell division and the
rapid growth of the bacterium. For the construction of
this shape-giving framework the bacteria use materials of
complex composition whose macromolecular organization is
quite different from any known polymer.
The cell walls of bacteria are composed of the fol
lowing basic building blocks: (a) amino acids, (b) sugars,
(c) amino sugars, (d) lipids, and (e) polyols. Two strik
ing chemical differences have been shown to exist in the
cell wall make-up of Gram-positive and Gram-negative
organisms. The cell walls of Gram-positive organisms have
little or no lipid material (0 to 2%) , while the cell walls
of Gram-negative organisms contain as much as 20% by
weight lipid material (29, p. 126}. The cell walls of
Gram-positive organisms also were found to contain a very
small number of amino acids, three or four for some, while
the Gram-negative organisms contain the whole range of
amino acids found in most protein hydrolyzates (71). The
chemical differences in the cell walls of Gram-positive
and Gram-negative organisms are represented in Figure 2.
Physically, the cell walls of Gram-positive organisms
are composed of one continuous layer bordered by the cyto
plasmic membrane. The Gram-negative cell walls consist of
several layers which surround the cytoplasmic membrane.
Figure 3, taken from the work of Salton (29, p. 23),
graphically represents this difference.
19
The cell walls of the Gram-positive organisms were
initially thought to be one homogeneous polymer. This
polymer in the course of its identification and character
ization has been given many names, for example, mucopeptide,
mucocomplex, mucoprotein, peptido-polysaccharide, glyco
peptide, glycosaminiopeptide, and murein (25, 29, p. 99-100).
For lack of a uniformly acceptable nomenclature glycosa
minopeptide will be used here. One portion of the Gram
negative cell wall amounting to 10 to 20% by weight has
been found to be a glycosaminopeptide in nature. More
recently, other polymers have been found to be part of the
Gram-positive cell wall in certain species. A group of
polymers called the teichoic acids were discovered by
Baddiley, Buchanan, and Carss (72). Their results were
published in 1958. Another polymer, which has not been
found as generally distributed as teichoic acid, is
Gram
Posi
tive
Gram
ega
tive
Cytoplasmic membrane
Amino Sugars: 10-18% Glucosamine, Galactosamine, Muramic acid
20
Total Reducing Substances: 40-50% Glucose, Galactose, Mannose, Arabinose, Rhamnose
Amino Acids: 40-50% ------~Glutamic Acid, Lysine or DAP,
Alanine, Glycine, Aspartic Acid, Serine
Total Lipids: 0-2% Phosphate: 0.2-0.8% Ribitol-5'-Phosphate
Cell Wall ~~Lipo-Glycosamino
Peptide ---------1
Cytoplasmic Membrane
Lipoprotein: 80% contains all Amino Acids
Glycosaminopeptide: 20% similar to that of Grampositive walls
Total Lipids: 16-20%
Figure 2. Chemical Composition of Bacterial Cell Walls.
21
c.w. m cyt. c.w. m cyt.
'. . -' ~ ...... . .
, , / ...
--..; .... ~ jllo
(a) (b)
Figure 3. Diagrammatic Representation of Cell Walls (29, p.23).
(a) Thick amorphous cell wall (c.w.) and underlying cytoplasmic membrane (m) and cytoplasm (cyt.) as found in Gram-positive bacteria.
(b) Multilayered cell wall and underlying membrane as found in Gram-negative bacteria.
22
teichuronic acid isolated by Janczura, Perkins, and
Rodgers (73) in 1961. Table 1 shows various groups of
substances found in the cell walls of both Gram-positive
and Gram-negative organisms. Some Gram-positive cell walls
may be comprised of 90% by weight of glycosaminopeptide
(e.g. Micrococcus lysodeikticus).
The cell walls of bacteria contain some unique chemi
cal compounds. Three of these are D-alanine, D-aspartic
and D-glutamic acids (74). The amino acids generally found
in nature are of the L-form (75). Two compounds,
diaminopimelic acid (DAP)and muramic acid are also found
only in cell walls. The structures of DAP and muramic
acid are shown in Figure 4. Work (76) first identified DAP,
while the structure of muramic acid was elucidated by
Strange (77) and Strange and Kent (78).
Since the discovery that the cell walls of Gram
positive organisms contained a relatively small number of
components (71, 79), much more time has been spent trying
to elucidate the structure of these cell walls than the
cell walls of Gram-negative organisms. The Gram-negative
organisms of the genus Spirilla, however, have been an aid
since it was found that their glycosaminopeptide layers
could be isolated in a pure form by treating the cell walls
with aqueous solutions of sodium dodecylsulfate (25).
Studies on the molecular structure of cell wall
glycosaminopeptide have advanced rapidly in the last few
years. Information has been gained from several sources,
Table 1. Principal Classes of Chemicals Found in
Cell Walls of Bacteria {29, p. 251).
Gram-positive
Gram-negative
Glycosarninopeptides Oligosaccharides Polysacchardies Teichoic Acids Teichuronic Acids Glycolipids
Glycosarninopeptides Proteins Lipids Polysaccharides Lipo-Polysaccharides Lipo-Proteins
23
COOH COOH
I I H N-C-H H-C-NH
2 I I 2 CH-2-cH2
a-E Diaminopimelic Acid (DAP)
NH 2
Muramic Acid
Figure 4. Structures of a-E Diaminopimelic Acid and Muramic Acid (76, 77, 78).
24
including the elucidation of the "Park nucleotides'' which
accumulate within the cell when some organisms are grown
in the presence of penicillin and other antibiotics. The
investigation of products from acid and lysozyme degraded
cell walls has also aided in the determination of the
glycosaminopeptide structure. Salton (80) made a major
discovery when he found a disaccharide in the dialyzable
fraction of a lysozyme degraded cell wall preparation. He
also found a similar compound in an acid hydrolyzate (81).
The nature of this and another dialyzable tetrasaccharide
component was reported by Ghuysen and Salton {82, 83).
25
The diasaccharide was found to be acetylglucosamine joined
by a 1-6 linkage to N-acetylmuramic acid. The tetrasac
charide was composed of two moles each of acetylglucosamine
and N-acetylmurarnic acid.
The postulated structures of these compounds are shown
in Figure 5. These data led Salton and Ghuysen (83) to
propose a structure for glycosaminopeptide with a backbone
of acetylglucosarnine and N-acetylmuramic acid with alter
nating 1-4 and 1-6 bonds. This proposed structure ~s shown
in Figure 6. In addition to these, some dialyzable
glycosaminopeptides of low molecular weight were also
found. These were separated by paper chromatography and
found to contain alanine, glutamic acid, lysine, glycine,
glucosamine and muramic acid. The amino acids were postu
lated to be in the peptide side chain (Figure 6) •
~CH2 0
H
H NHCOCH 3 0
I CH 3CHCOOH
0
H ~--' H H
NHCOCH 3 \ H NHCOCH 3
CH 3CHCOOH
H
H
(a)
CH 3CHCOOH \ 0
(b)
26
Figure 5. The Proposed Structure of Amino Sugar, Di-and-Tetrasaccharides from Micrococcus _lysodeikticus (82) .
(a) Amino sugar disaccharide isolated from lysozyme digestion mixture.
(b) Amino sugar tetrasaccharide isolated from lysozyme digestion mixture.
(1-+6)
~Backbone Structure~
(1-+4) (1-+6) (1-+4) (1-+6) (1-+4) (1-+6)
-AG-AMA AG AMA AG :AMA--AG AMA--
Peptide Peptide
Figure 6. Type of Molecular Structure Proposed for the Cell Wall of Micrococcus lysodeikticus (83).
This figure shows the arrangement of side chains on an acetyl amino sugar backbone possessing alternating 1-+4 and 1-+6 bonds between N-acetyl-muramic acid (AMA) and N-acetyl-glucosamine (AG).
I'V -...]
28
Park (84) in 1952 found that a number of nucleotides
accumulated in the cells of organisms grown in the presence
of penicillin. The structure of the "Park nucleotides",
when elucidated, gave an indication of the structure of
the peptide chain proposed by Salton and Ghuysen (83)
(Figure 6). A structure of one of the "Park nucleotides"
which is thought to be a precurser to glycosaminopeptide
is given in Figure 7. These data can be used to infer the
structure of the peptide chain, as well as its linkage to
the disaccharide shown in Figure 6.
Ghuysen (85) isolated two glycosaminopeptides from the
lysozyme-digested cell walls of Micrococcus lysodeikticus.
Each of the compounds had the same molar proportions of
components. For each mole of glutamic acid found in the
glycosaminopeptide there were found 2 moles of disaccharide,
2 moles of alanine, 1 mole of glycine and 2 moles of lysine.
These two glycosaminopeptides were separated by electro
phoresis. Treatment of the glycosaminopeptides with an
enzyme, Streptomyces F2B amidase, liberated a disaccharide
similar to the one first described by Salton and Ghuysen
(83) (Figure 6) and a peptide. The new N-terminal group
of the peptide was alanine. Therefore, alanine was bonded
directly to the muramic acid through an amide bond just as
in the "Park nucleotide" (Figure 7). Salton (86) has also
shown that alanine is the amino acid linked to the muramic
acid found in the cell walls of several organisms.
lr~~ o- o-H-c-c-c-c- I I I I CH20-P-O-P = 0
H H ~ b I
Uridine - 5' Diphosphate
0
NH p I HC - CH C=O I 3 I I
C = 0 CH3 : 1-- ---------------- ~ NH I
I : HI - CH3 I
C = 0 I ,_ -- ________________ ., NH I
I I
TH- (cH2> 2 c = o :
~~QH __ - -----1----- ----~ I NH
Muramic Acid
L - Alanine
D-Glutamic Acid
I HC -I
L-Lysine {CH 2 ) 3-c,H2
I C = :--------!H 0 NH2
D-Alaninei H!- (CH3 I c = 0
.. -------I I NH
: I D-Alanine: HC - CH 3
I I L -- - - - - COOH
Figure 7. Structure of a "Park Nucleotide" from Staphylococcus aureus (25} .
29
Primosigh et al. {87) found two low molecular weight
glycosaminopeptides in digests of the rigid portion of
Escherichia coli cell walls. Pelzer (88) analyzed the
structures of these compounds. The structures are shown
in Figure 8. The amino acid make up differs from that
found in Figure 7, in that lysine has been replaced by
diaminopimelic acid.
30
On the basis of the compounds isolated and studied bY
Ghuysen (85) and on hydrazinolysis experiments, Salton (86)
proposed a model for the cell walls of Micrococcus lyso-~
deikticus. This model is shown in Figure 9.
According to Salton (29, p. 146-147), Schacher, Bayley,
and Watson have suggested the hypothetical glycosaminopep
tide unit shown in Figure 10 as being the basic cell wall
unit of Aerobacter cloacae.
Staphylococcus aureus from which the 11 Park nucleotide"
shown in Figure 7 was isolated, has a cell wall composed of
two polymers, glycosaminopeptide and teichoic acid. The
structures of four basic types of teichoic acid are shown
in Figure 11. The cell walls of Staphylococcus ~
contain Type b teichoic acid. Mandelstam and Strominger
(89) have carried out further investigations on the
teichoic acid and glycosaminopeptide fractions of
Staphylococcus aureus and have suggested a possible mode
of teichoic acid glycosaminopeptide attachment. This mode
Of attachment is shown in Figure 12. Since glycine was not
found in the "Park nucleotides 11 these investigators proposed
31
GnAc GnAc - lactyl GnAc GnAc - lactyl
I I L -ala L -ala
I I D -glu D -glu
I {COOH)
I meso DAP meso -DAP
I Compound c5 D -Ala (COOH)
Compound c6
Figure 8. Structure of Two Glycosaminopeptides Isolated from Escherichia coli Cell Walls (88).
The glycosaminopeptides were isolated from the rigid layer of lysozyme digested cell walls of Escherichia coli B.
R - GnAc - GnAc --- Rl
I lactyl
I ala I
ala I
glu
I lys (- e:NH 2 ) I
gly I
( e:NH-) - gly (- COOH) lys I
glu I
ala I
ala I
lactyl
I 111 R - GnAc - GnAc -
32
Figure 9. Hypothetical Glycosaminopeptide Subunit of the Cell Wall of Micrococcus lysodeikticus (86).
Ala
Glu
DAP
Ala
H NH I
O=C I CH 3
H
0 H
~TH C=O
r------------1 I NH I I I CH-CH3 I I I C=O 1------------1 I NH
H
0
33
NH 2 COOH---, I I I
HOOC-CH-(CH) -CH IDAP 2 3 I I
NH : 1-------4 C=O I I I
HOOC-(CH 2) 2-CH :Glu I I NH I
1------..f C=O I I I
CH 3-CH 1Ala I :
NH I 1----- __ J
C=O I
CH3"CH if 0
II NH-C-CH 3
I I I yH-(CH2)2-COOH
I C=O ~-----------1 I NH NH2 : I I
TH-(CH2)3-CH-COOH
I C=O 1-----------1
NH I CH-CH 3
I I ""-- - - - - - -- COOH
Figure 10. Hypothetical Subunit of the Cell Wall of Aerobacter cloacae (29, 147).
34
(a) ~lanyl - glucosyl - ri~ito~ L O=P-OH I n
(b) ~lanyl - N - acetylglucosaminyl - ribttoll
L O=P-OHJ I n
(c) [
lanyl - glycefolJ
O=P-OH I n
(d) ~anyl - glycosyl - gly,ero~ L O=r-OHJ 0
Figure 11. Four General Types of Teichoic Acid (29, p. 159).
- GnAc - GnAc - GnAc - GnAc -
I I lactyl lactyl
I I L-ala L-ala
I I ~ /D-glu '\ ~,..........D-gllu , rl\.i'\ ~
~ '? I l~\.i -J ~,~ ~"i. L-lys,.......... L-lys
/'--~ I D-ala
I D-ala
D-ala I
D-ala I
GnAc GnAc GnAc I
GnAc I I
-Ribitol - P - Ribitol - P - Ri~itol (a)
- P - Ribitol - P -
- GnAc - GnAc - GnAc - GnAc -I I
lactyl lactyl I I
L-ala L-ala / I :i" '? I ).1" 1-
..........-o-glu '\.~-...;. 'o-glu '\.~
=1"< I / I/ '\.~-...;. L-lys L-lys / I I
D-ala D-ala I I
D-ala D-ala
I I GnAc GnAc GnAc
"" I ~ GnAc
R.l. - ~u~tol - P - Ribitol - P - Ribitol - P -
(b)
Figure 12. Proposed Structure of Cell Walls of Staphylococcus aureus (89).
35
In representation a, the linkage between and glycosaminopeptide is not specified. b the teichoic acid is shown attached to tide.
the teichoic acid In representation
the glycosaminopep-
36
that glycine served as a peptide bridge between the peptide
side chains of the glycosaminopeptide.
There are still many questions concerning glycosamino
peptides and cell walls which have not been answered. For
example, the mode of teichoic acid attachment, the mode of
oligo-and polysaccharide attachment, the precise sequence
of the amino acids in the peptide side chains, and the sig
nificance of the undialyzable fraction of lysozyme digested
cell walls are not known. However, some common structural
properties have been established in all the studies of
glycosaminopeptides. They possess a glycosidically-linked
backbone composed of alternating units of N-acetylglucosa
mine and N-acetylmuramic acid and the peptides are linked
through the amino group of alanine and the carboxyl group
of muramic acid. It is now known that the chemical com
ponent responsible for the rigidity of cell walls in both
the Gram-positive and Gram-negative species is glycosamino-
peptide (29, p. 8, and 81).
Some data relating the qualitative and quantitative
aspects of the amino acid analyses of bacterial cell walls
are given in Tables 7, 8, and 9, p. 79, 82, 84 of subsequent
sections.
To analyze the components of the cell wall, the cell
wall itself must be isolated. The isolation of the cell
wall generally follows the sequence: (a) separation of
the cells, (b) pretreatment of the cells, (c) disruption of
the cells, (d) separation of the cells, cell walls and cell
components, and (e) purification of the cell walls.
37
The general procedure for collection and concentration
of the cells is centrifugation of the cell suspension. The
pretreatment consists of washing with salt or buffer solu
tion, or distilled water. If the cells are pathogenic they
can be killed before washing. Some organisms which clump
may be pretreated with surfactants to reduce aggregation.
The cells may be disrupted by several methods (29,
p. 42-63) : {a) autolysis, {b) osmotic lysis, {c) heat
treatment rupture, and {d) mechanical disintegration.
Autolysis is a process in which the organism is placed
in a solvent and allowed to lyse. The disadvantage of this
process is that some bacteria contain lysozyme-like enzymes
which actively destroy their cell walls. If the solvent
used does not immediately kill the organism, the isolated
cell walls may be depleted of certain chemicals due to the
utilization of the cell wall by the starved organism. There
is, therefore, some doubt that the cell wall material iso
lated in this manner is completely intact.
Osmotic lysis is a process in which the organism is
grown in a medium of high solute concentration. The medium
is then rapidly diluted and this causes the cell wall to
rupture. This method works very well with halophilic
organisms.
Heat treatment rupture is a process in which the
organisms are added to hot water for a short period,
thereby causing the organism to rupture. This procedure
has not been found applicable to Gram-positive organisms
(29' p. 42-63).
38
Mechanical disintegration consists of a group of
processes in which the cell walls are physically and
mechanically ripped apart. Mechanical disintegration is
the most versatile method of disruption. There are several
methods of mechanical disintegration: (a) grinding, violent
aggitation or compression with abrasives such as sand, glass
beads, steel balls or alumina; (b) sonic or ultrasonic dis
integration; (c) decompression rupture; and (d) pressure
cell disintegration.
After the cells are ruptured, the cell components, the
cell walls, and the unruptured cells must be separated.
The separation can be made by differential centrifugation
or by density gradient sedimentation (90).
Differential centrifugation is centrifugation at dif
ferent speeds in order to separate particles of different
size. The unruptured cells are separated by centrifugation
at relatively low speed. The supernatant of this separa
tion is decanted into another centrifuge tube and the cell
walls and cell components are separated by centrifuging at
a higher speed. A scheme for this type of separation is
given in Figure 13.
Density gradient sedimentation is a separation carried
out by placing the cells, cell walls, and other cell com
ponents in a layer over a solution which has been carefully
placed in a centrifuge tube. The solution in the centrifuge
39
DISINTEGRATE CELL SUSPENSION
Deposit {Intact cells}
Discard
Centrifuge
2,000 - 3,000 xg; 5-10 minutes
Supernatant (cell walls, cytoplasm)
Centrifuge
5,000 - 9,000 xg
20 minutes
Deposit {crude cell wall fraction)
Supernatant (cytoplasmic
material)
Figure 13.
Discard
General Scheme for Differential Centrifugation (29, p. 58).
40
tube is then added in a manner such that it has a density
gradient. Sucrose and glycerin are the solutes generally
used to produce the gradient. After the layer of cellular
material is placed on top, the tube is centrifuged for
several minutes. This causes the cells, cell walls, and
cell contents to separate into three separate layers which
can then be removed.
The cell walls may be purified by several methods.
The first and probably the best method is repeated washing
with distilled water. Salton (29, p. 42-63) recommended
the use of sodium chloride solutions, while others have
used various buffers. Cummings and Harris (79) have used
a procedure in which they digested the cell walls with
trypsin and ribonuclease followed by digestion with pepsin.
This procedure may or may not remove antigens and other
surface components from the cell wall.
41
III. EXPERIMENTAL
A. MATERIALS AND METHODS
Cultivation of Cells. The strain of Thiobacillus
thiooxidans used for this study was culture number 8085,
American Type Culture Collection (ATCC), 2122 M Street, N.W.,
Washington, D. C.
After the culture obtained from ATCC was growing well,
several 5 ml samples were taken. These samples were
maintained in a cold room (4 C) in a liquid state, and were
used to start a new stock culture periodically. The stock
culture in use at the time was routinely transferred to a
fresh medium once every seven days. In either case a flask
containing 100 ml of medium was inoculated with 5 ml of
inoculum from the stock culture.
The medium employed in this work was as follows:
(NH4 ) 2so4 0.2 g
MgS04 ·7H20 0.5 g
CaC1 2 0.25 g
KH2Po4 3. 0 g
FeS04 trace (0.01) g
Sulfur 10.0 g
H20 1000 ml
To prepare the medium, the salts were dissolved in the
water. The medium without sulfur was sterilized for 30
minutes in an autoclave (Rectangular type 24x36x48 inches,
42
American Sterilizer Co., Erie, Pa.) at a temperature of
120 C and pressure of 15 PSIG. The sulfur was added with
out sterilization, just before the medium was inoculated
with organisms. This was done because sulfur was found to
form an amorphous mass on sterilization, thus reducing the
exposed surface available for oxidation by the organism.
The pH of the medium was in the range of 4.0 to 4.8.
The cells were grown in a 16 liter fermentor described
by Crum (91). An over-all sketch of the apparatus used is
given in Figure 14. All cell growth took place at ambient
temperature conditions which ranged from 21 to 28 C. The
cells were grown in the medium described previously. Growth
was started by inoculating the previously sterilized fer-
mentor with 270 ml of stock culture. The stock culture
had been allowed to grow for seven days on a rotary shaker
{Model CS-62630, New Brunswick Scientific Co., New
Brunswick, N. J.). The incubation was stopped after eight
days. Experiments were run to determine if surfactants
would aid in the growth of the organism. These are
described in a following section of this chapter.
The growth of T. thiooxidans was followed by titration
of the sulfuric acid formed by the organism. Titrations
were performed in the following manner. An appropriate
volume of medium {usually 10 ml) was taken, filtered, and
the residue washed with four volumes of deionized water.
The combined filtrate and washings were then diluted to
Thermometer Well1 ....
Motor
..<"rio
Sample Port Ferrnentor
'"""
0 0 0 0 0 0 0 0
0 0 0
I I I
/-/---+-' Agitator z Air
Sparger
Air Filter
pH Electrodes
l
pH Meter
Pressure --------t Regula tor
t I I Rotameter
Figure 14. Sketch of Fermentation Apparatus (91).
Air Supply
~
w
44
approximately 150 ml and titrated with 0.1 N sodium hy
droxide using phenolphtalein as the indicator.
The cells grown in the fermentor were harvested by
continuous centrifugation (Model LCA-1, High Speed Centri
fuge, Lourdes Instrument Corp., Brooklyn, N.Y.) at 4,250
to 4,500 x g .
Cells which were not to be used immediately were
stored in the frozen state.
Preparation of Cell Walls. Two pretreatment pro-
cedures were employed before the cells were ruptured. The
first pretreatment, which was used in all cases, consisted
of washing the cells three times with deionized water. If
the cells were stored in the frozen state for any length
of time, they were again washed three times with deionized
water before they were used. The second pretreatment,
which was evaluated experimentally but not used in routine
work, was that described by Suzuki and Werkman (57). This
pretreatment consisted of treating the cells in solution
with two ion exchange resins. The procedure was as follows:
To the cell suspension of 10 to 20 mg of washed cells per
ml (dry weight) in deionized water was added 2 g of AG-1X2
(Bio-Rad., 32nd and Griffin Streets, Richmond, Calif.) and
2 g of IR-120 (Bio-Rad.) ion exchange resins. The cell
suspension and ion exchange resins were thoroughly mixed
and placed on the rotary shaker for 20 minutes. The
mixture was filtered to remove the ion exchange resins,
and the cells ruptured in the usual manner.
45
The cells were ruptured by two methods; extrusion under
high pressure through a small aperature and sonication.
The procedure for the extrusion rupturing was as fol
lows: The pretreated cells were suspended in enough buffer
or deionized water to give a suspension of 10 to 20 mg of
cells per ml (dry weight) . The cell suspension was placed
in the precooled (4 C) compression chamber (Catalogue
No. 4-3398 American Instrument Co., Inc., Silver Springs, Md.).
The compression chamber was placed on a press (Model No. 12-
105 Wabash Metal Products Co., Wabash, Ind.), and the cells
were forced out at a pressure of 16,000 to 20,000 PSIA by
regulation of the control valve on the compression chamber.
The cell suspension was collected in a small beaker sur
rounded with ice. In most cases the cells were returned to
the compression chamber and expressed as above a total of
three times. However, some samples were tested after going
through the process only once.
The procedure for sonication was as follows: The pre-
treated cells were placed in a 100 ml Rosett cooling cell or
a 50 ml polycarbonate centrifuge tube. Enough buffer or
deionized water was added to give a suspension with 10 to
20 mg of cells per ml (dry weight). The cell suspension,
in its receptacle, was lowered into an ice water bath. The
suspension was sonified (Model 575, Sonifier, Branson Sonic
Power, Division of Branson Instruments Inc., Danbury, Conn.)
for the specific time interval required at a power input of
6.6 to 7.1 amperes. When samples were taken for protein
determination, they were taken from the supernatant after
the entire cell suspension had been centrifuged at 13,000
~ g for 10 minutes. Enough buffer or deionized water was
then added to the system to bring it back to its original
volume to keep the volume constant.
The whole cells, cell walls, and cell components were
separated after cell rupture as follows: ~he whole cells
were removed by centrifuging at 2,100 x g for 10 minutes.
The supernatant was then centrifuged for 20 minutes at
8,400 x g. The sedimented cell walls were saved for
further purification by linear sucrose density centrifuga-
tion.
46
Linear sucrose density centrifugation was used to
further purify the crude cell walls obtained by differen
tial centrifugation of the ruptured cells. The apparatus
used is shown schematically in Figure 15. After the rubber
tubing was clamped, 25 ml of sucrose (40gper 60 ml of
1 M potassium chloride solution) and 25 ml of 1 M potassium
chloride solution were added to chamber A and B respec
tively. All bubbles were forced out of the rubber tubing
and the air was turned on. After the clamp was opened,
about 5 ml of the solution in chamber A was drawn into a
small beaker through a 2.5 mm (outside diameter) plastic
tUbe. The beaker was replaced with a 50 ml polycarbonate
centrifuge tube and about 35 to 40 ml of solution was
carefully drawn from chamber A in a manner which allowed
47
Air B
T 9"
Plastic Tube
Clamp
Figure 15. Device used to Produce Linear Sucrose Gradients.
48
stratification. The clamp between chambers A and B was
closed and the solution remaining in chamber A was used to
suspend the cell walls to be treated. This was accomplished
by sonication at about 2 amperes for 30 seconds. The sus-
pended cell walls were carefully layered on the surface of
the sucrose gradient. The centrifuge tube was then care
fully placed in an International horizontal rotor centri-
fuge (Model CL No. 50204 H International Equipment Co.,
Boston, Mass.}, and centrifuged at 1,500 to 1,600 x g for
60 to 90 minutes. Generally only two layers were observed,
however, sometimes three layers were found when doing the
initial linear sucrose gradient. The separate layers
formed are described in Figure 16. The cell wall layer was
removed with a 50 ml syringe with a size 15 cannula. The
same procedure was repeated a second time.
The cell walls after two linear sucrose centrifuga
tions were washed three times with deionized water, three
times with 1 M sodium chloride, and then three more times
with deionized water.
Cell walls were tested for contamination by intra-
cellular material by measuring their absorption of light
in the ultraviolet range. The spectrophotometer used was
a Beckman DK-2A Automatic Scanning Spectrophotometer
(Beckman Instruments, Inc., 2500 Habor Blvd., Fullerton,
Calif.).
Cell not to be used immediately were walls which were
stored in the frozen state (-15 C)·
(a)
Figure 16. Layers Formed During Linear Sucrose Gradient Purification-
(a) Precipitate of intact cells. (b) Yellow Phase. (c) Cell wall layer.
49
Cell Wall Components. The components of the cell
wall were identified by hydrolysis followed by paper
chromatography or amino acid analysis. Some cell walls
were digested with trypsin preceding hydrolysis.
For the determination of the amino acid content of
50
the cell walls, the walls were hydrolyzed in 6N hydro
chloric acid for various periods of between 12 and 24 hours.
The cell walls at a concentration of 100 mg per ml (wet
weight) and the hydrochloric acid were sealed in a pyrex
tube for digestion. The sealed glass tubes were inserted
into a 3/4 x 8 inch pipe with nipples on both ends, and
the pipe was placed in a convection hot air oven (Model 16,
Precision Scientific Co., Chicago, Ill.) at 103 to 107 C.
After the selected time had elapsed, the tube was removed
from the oven, cooled and opened. The residue in the tube
was filtered through filter paper and washed with deionized
water. The filtrate and washings were evaporated to
dryness on a Rinco (Model 1007-4 Rinco Instrument Co.,
Greenville, Ill.) evaporator. The residue was dissolved in
deionized water and the mixture evaporated to dryness. This
procedure was repeated two more times.
Cell walls for sugar and polyol analysis were hydro-
lyzed by two different methods. The first method was
hydrolysis for two hours in 2N sulfuric acid at 100 to
103 C. This digestion was also carried out in a sealed
PYrex tube as described above except that an oil bath
51
maintained at a temperature of 100 to 103 c was used rather
than the hot air oven. The digested material was adjusted
to pH 4.5 with solid barium hydroxide. The barium sulfate
and other insoluble material formed was removed by centri
fugation. The supernatant was taken nearly to dryness on
the Rinco evaporator.
The second method was digestion in the presence of
IR-120 ion exchange resin. About 1 g of wet resin and
about 0.1 g of wet cell walls along with 3.5 ml of water
were added to a pyrex tube. The test tube was sealed, and
the contents digested for 24 hours at 120 C in the hot a1r
oven. The residue was filtered and the filtrate taken to
dryness on the Rinco evaporator.
The cell walls to be digested with trypsin were placed
in a pH 7.9 buffer solution containing 0.5 mg per ml of
trypsin. Approximately 20 mg of wet cell walls were added
per ml of buffer solution. The cell walls and buffer were
thoroughly mixed in an erlemeyer flask. The flask was
placed in a shaking, constant temperature bath (Model RW
150 C, New Brunswick Scientific co., New Brunswick, N.J.),
and the cell walls digested for two hours at 35 C. The
residue was separated by centrifugation and washed once
With buffer and three times with deionized water. The
wash solutions were discarded and the residue, which con
tained the treated cell walls, was digested with 6 N hydro
chloric acid in a sealed pyrex tube as described previously.
52
Paper chromatograms for amino acid detection were
prepared by the two-dimensional ascending method. The paper
used was 11 by 11 inch Whatman No. 1 filter paper. The
first solvent used was butanol, acetic acid, and water
(120:30:50:vol./vol.}. The second solvent was phenol,
water, and concentrated ammonia (182:18:l:vol.jvol.). The
amino acids were located by spraying with ninhydrin in
acetone. The ninhydrin spray was prepared by mixing 2 g of
ninhydrin with 100 ml of acetone.
The amino acids were identified by comparing their Rf
values with those from papergrams prepared with solutions
of known compounds, and by adding known samples along with
the unknown and determining if the spots overlapped. The
Rf values were defined as: the length the sample moved
divided by the length the solvent front moved. If known
solutions were not available, Rf values were compared
with those given by Smith (97).
The sugars and polyols were separated by single dimen-
sional descending paper chromatography. The paper used was
11 by 22 inch Whatman No. 1 filter paper. The solvent used
was butanol, acetic acid, and water. The proportions were
the same as given previously. The sugars were located by
spraying with either periodate or silver nitrate spray
reagents. Silver nitrate spray was prepared by adding 0 · 1
ml of saturated silver nitrate solution to 100 ml of
acetone. After spraying the papergram with silver nitrate
spray reagent, it was dried and then sprayed with a O.S%
53
solution of sodium hydroxide in ethanol. Sugars gave brown
or black spots with this treatment.
Both sugars and polyols can be located with periodate
spray. The periodate spray was prepared by mixing 1 ml of
periodic acid (2.28 g in 100 ml of deionized water) and
19 ml of acetone. The periodic acid-acetone mixture was
sprayed on the dry papergram and it was allowed to stand
for 3 to 4 minutes. The paper was then sprayed with a
benzidine solution prepared by mixing 184 mg of benzidine
in 0.6 ml of acetic acid, 4.4 ml of water, and 95 ml of
acetone. Sugars and polyols gave a white spot on a blue
background.
Sugars and polyols were identified by comparing their
Rg values to those of known samples run in a similar manner.
The R values were defined as: the length the sample moved g
divided by the length the glucose sample moved.
The quantitative amino acid analyses of digested cell
walls were performed by the Experiment Station Chemical
Laboratories of the college of Agriculture at the University
of Missouri, Columbia, Missouri.
The samples were prepared by digestion, filtration, and
washing as already described. The filtrate and washings
were collected in a previously weighed glass ampule. The
digestion mixture was taken to dryness and water was added.
This operation was repeated four times. The dry samples
were then stored in a dessicator overnight. After drying
54
in the dessicator the ampule was again weighed. The ampules
were sealed after weighing and stored in the frozen state.
The following procedure was used by the Experiment
Station Chemical Laboratory. The samples were dissolved in
10 ml of 0.1 M hydrochloric acid, and 4 ml samples were
chromatographed on a Technicon Amino Acid Analyzer.
Protein and Nitrogen Determinations. Protein and
nitrogen determinations were used to measure the amount of
cell rupture produced during rupture experiments.
Protein was determined by precipitation with trichlo
roacetic acid (TCA) , and the precipitated protein measured
by the biuret method (93) as follows: The sample was added
to a 15 ml graduated centrifuge tube. A volume of 50% TCA
equal to approximately one quarter the sample volume was
added, and the mixture was brought to at least 3 ml with
10% TCA. The suspension was mixed and allowed to stand
for 5 to 15 minutes. The supernatant was drawn off leaving
0.2 to 0.3 ml. With the aid of a spatula the precipitate
was thoroughly mixed with the remaining liquid. Eight ml
of biuret reagent was added and the solution and precipi
tate mixed until the precipitate was completely dissolved.
The solution was brought to 10 ml and allowed to stand for
30 minutes. After this period the optical density of the
samples were measured at 540 m~ in a Bausch and Lomb
Spectronic 20 Colorimeter (Bausch and Lomb, Incorporated,
Rochester, N. Y.). A blank was prepared in a similar
m A standard curve was prepared anner using deionized water.
55
using a solution of bovine serum albumin (BSA). To prepare
the biuret reagent the following procedure was used: 1.50 g
of hydrated cupric sulfate (Cuso 4 .sH20) and 6.0 g of sodium
potassium tartrate (NaKC 4H40 6) were weighed and transferred
to a dry one liter volumetric flask. The salts were dis
solved in 500 ml of previously boiled deionized water.
Three hundred ml of 10% sodium hydroxide, prepared from a
30% stock solution diluted with boiled deionized water, was
added with constant swirling. The solution was then
brought to one liter with boiled deionized water. The
biuret reagent was stored in a polyethylene bottle under
refrigeration (4 C).
The second means of protein determination was a spec-
trophotometric method outlined by Seaman (94) . The sample
was diluted until an optical density reading could be taken
at 260 m~ with a Hitachi-Perkin Elmer, Model 139, Spectro
photometer (Coleman Instruments corporation, Maywood, Ill.)·
The optical density was then measured at 280m~. The ratio
of the optical density at 280 m~ to the optical density at
260 m~ was used to determine a factor, F, given by Seaman.
The amount of protein was calculated as follows: mg pro
tein/ml = F x optical density at 280 m~ x dilution.
The soluble nitrogen in the supernatant of samples
centrifuged at 13,000 x g for 10 minutes was used as a
m 1 t Nitrogen was deter-easure of the degree of eel rup ure.
mined by the Kjeldahl method and the procedure employed was
as follows: To each clean dry 100 ml Kjeldahl flask was
56
added 1.5 g of potassium sulfate. Then enough liquid
sample was added to supply from 0.6 to 6.0 mg of nitrogen.
To each flask 1.5 ml of mercuric sulfate solution (prepared
by diluting 12 ml of concentrated sulfuric acid to 100 ml
and adding 10 g of red mercuric oxide) was added. Three or
four glass beads and 3 ml of concentrated sulfuric acid
were added to each flask. The flasks were heated on a
Kjeldahl digestion rack (Laboratory Construction Co.,
Kansas City, Mo.) until the water had been boiled off and
white sulfur trioxide fumes were no longer formed. The
solution at this point was completely clear. The clear
solution was then boiled for 30 minutes and allowed to cool.
Twenty-five ml of distilled water was added to each flask,
and the flasks were again cooled. Fifteen ml of 13 N sodium
hydroxide was then carefully added to the Kjeldahl flask
in an ice bath. The sodium hydroxide formed a layer on the
bottom of the flask. After the sodium hydroxide was added
the flask was connected to the Kjeldahl distillation ap
paratus with as little mixing as possible. At this point a
boric acid trap was placed on the condenser of the distilla
tion apparatus. The boric acid trap was prepared by adding
20 ml of 2% boric acid and two drops of Tashiro's indicator
to a 125 ml erlenmeyer flask. The Tashiro's indicator was
prepared by adding 0.25 g of methylene blue and 0.375 g of
methyl red to 300 ml of 95% ethanol. The tip of the
condenser was placed as far as possible below the surface
of the boric acid. The digested mixture and the sodium
hydroxide were then mixed and the solution was boiled for
20 to 25 minutes. At this point salt had started to form
in the digestion flask. The boric acid bath was removed
and the condenser tip washed down with 3 ml of distilled
water before the flask was taken off the distillation
57
apparatus. The ammonia trapped in the boric acid solution
was titrated with 0.02 N sulfuric acid. The Tashiro's
indicator turned from green to grey at the end point. Each
ml of 0.020 N sulfuric acid was equivalent to 0.28 mg of
nitrogen.
Surfactant Studies. Tergitol 08 and TWeen 80
surfactants were tested to see if they would enhance the
growth of T. thiooxidans. TWeen 80 was supplied by Atlas
Chemical Industries, Inc., Chemicals Division, Wilmington,
Del. and Tergitol 08 by Union carbide Corporation, South
Charleston, W. Va.
Initial tests for foaming were obtained by passing
air through a fine capillary tube into the culture medium
containing sulfur in a 500 ml erlenmeyer flask. The amount
The medium of foam resulting was visually observed.
containing surfactant was also tested by placing it on the
rotary shaker and shaking for various periods, and then
tested for foaming in the described manner.
tests Were made using TWeen 80 as a Two fermentation
wetting agent. These tests were made in the same manner as
those described under "Cultivation of Cells" (p. 41 > with
58
the exception that Tween 80 was added after the medium had
been sterilized and before the sulfur was added. The tests
were performed in a two liter capacity fermentor described
by Li (92) which allowed visual observation.
I t I ·~
'
59
B. RESULTS
Surfactant Studies. Two surfactants, Tergitol 08 and
Tween 80, were examined to see if they would reduce the lag
period found in the growth of the organism Thiobacillus
thiooxidans.
From visual observation it was found that Tergitol 08
1n concentrations of 50 to 500 ppm partially wet the sulfur
in the medium and caused it to be more evenly distributed
when the medium was incubated on the rotary shaker for a
period of seven days. However, some sulfur remained on the
surface, when the medium was aerated for a short period.
The sulfur rose to the surface and floated on a layer of
foam. This problem did not occur with Tween 80. The
foaming effects of TWeen 80, as visually evaluated, seemed
minimal.
Experiments were performed to determine the effect of
Tween 80 concentration on growth. The growth of the
organism was followed by measuring the formation of acid.
The study was performed in 500 ml erlenmeyer flasks incubated
on the rotary shaker. The data from this study are given
in Table 2 and plotted in Figures 17 and 18. Figure 18
gives the data for the samples containing 12.5 ppm TWeen 80
and for the sample containing no TWeen 80 expanded near the
origin. f tor constructed by
Two tests were made in a glass ermen
Ll. (g 2). 'th TWeen 80 concentrations These tests were made W1
Of 1 lt e g iven in Table 3 and 2.5 and 50 ppm. The resu s ar
Table 2.
Incubation 0 -Time (Hours)
0 3.9
24 6.0
60 13.9
84 20.2
108 26.9
168 41.0
288 57.0
432 65.1
The Effect of Surfactant Tween 80 on the Growth
of Thiobacillus thiooxidans. Shake Flask Studies. -
Tween 80 Added (ppm)
12.5 50 100 -Sulfuric Acid Formed (MEQ./100 ml}
3.8 3.7 3.4
6.4 5.9 5.5
14.8 14.2 11.9
21.1 19.5 17.4
26.6 25.5 24.0
40.6 39.0 35.6
55.6 54.7 54.6
64.1 62.5 62.9
230
3.4
4.0
5.4
8.6
10.3
23.6
43.6
51.2
0'1 0
70
60
~
...:I ::E: 0 50 0 r-4 ......... . 0
~ 40 0 IJ:l
I I I ~ 0 no wetting agent 0 ~'>.!
30 i I I i Ill 0 no wetting agent 0
0 12.5 ppm Tween 80 H u
20 j I I e 230 ppm Tween 80 l ~ 0 50 ppm Tween 80 ~
u H 1 f7/ e 100 ppm Tween 80 ~ :::J ~'>.! ..:I :::J tl)
10
0 0 4 8 12 16
TIME OF INCUBATION (DAYS)
0 20 4 8 12 16 TIME OF INCUBATION (DAYS)
20
F'iqurc 17. The F.ffect of Surfactant Tween 80 on the l.irowth of Thiohacillus thiooxidans. Shake Flask Studies. 0'1 1-"
30
~ ~
0 0 r-1 '-. ()I li:l ~ -0 ri:j
~ 0 r:t..
0 H t) ~
t) H p:; :::::> r:t.. ....::1 :::::> 5 U)
0 0
Figure 18.
62
0 no wetting agent added
• 12 ·5 ppm Tween 80
60 80 100 20 40
120
TIME OF INCUBATION (HOURS)
The Effect of Surfactant TWeen 80 on the Growth of Thiobaci11us thiooxidans. Shake Flask Studies.
Table 3. The Effect of Surfactant Tween 80 on the Growth of
Thiobaci11us thiooxidans. Fermentor Studies.
No Tween 80 Added 12.5 EEm Tween 80 Added 50 ppm Tween 80 Added Incubation H~SO' Formed Incubation H~SO' Formed Incubation H2so' Formed Time (Hours) { EQ 100 m1) Time (Hours) ( EQ 100 m1) Time (Hours) (MEQ 100 m1)
0 2.6 0 2.6 0 1.8
24 2.7 12 3.0 8 1.7
38 3.0 24 3.6 23 2.0
49 3.5 34 4.0 35 2.1
61 5.6 46 7.3 47 2.6
82 11.6 58 9.5 60 2.6
98 21.8 70 11.1 80 2.7
109 28.0 80 12.2
122 30. 3 94 12.8
144 36.2 106 13.2
168 38.7 118 13.9
191 41.8 141 14.7
"" 217 45.9 185 16.2 w
and Figure 19. For comparison, the data from a test using
no surfactant are also included in Figure 19.
Cell Rupture. Two methods were employed to rupture
64
the cells. They were sonication and pressure cell rupture.
Sonication was performed by exposing the suspended cells to
ultrasonic waves which ruptured the cell walls. For
pressure cell rupture the suspended cells were compressed to
a high pressure and forced through a small orifice. In
addition to washing with deionized water, some cells were
pretreated by contacting them with ion exchange resins. The
rupture of cells was studied in deionized water and in a
basic buffer (pH 7.9) solution. The results of these
experiments are summarized in Table 4.
The per cent soluble nitrogen given in Table 4 was
defined as the ratio of the Kjeldahl nitrogen in the super
natant centrifuged at 13,000 x g for 10 minutes to the total
Kjeldahl nitrogen in the sample before treatment.
The paper chromatograms for the Paper Chromatography.
detection of amino acids were prepared by the two dimensional
ascending method. The amino acids were located by spraying
with ninhydrin. The solvents used were butanol, acetic
acid, and water (120:30:50:vol./vol.) and phenol, water, and The amino acids
concentrated ammonia (182:18:l:vol./vol.) •
Were identified by comparing Rf values with those from
papergrams prepared with known solutions and by adding
known samples along with the unknowns and determining if
the spots overlapped. The Rf values for those amino acids
-..:I ::E: ..........
• 01 ~ ~ -0 ~
~ 0 ~
0 H 0 F:l! u H ~ ::::::> ~ H :::> til
50
I I
40
30
20
' 10
0
0
no Tween 80 added ~
I - 12.5 ppm ~ween 80 added
50 ppm Tween 80 added
40 80 120 160 200 240
INCUBATION TIME (HOURS)
Figure 19. The Effect of Surfactant Tween 80 on the Growth of Thiobaci11us thiooxidans. Fermentor Studies.
0'1 l11
Table 4. Thiobacillus thiooxidans Cell Rupture Experiments.
Test I Test II Test III
Type of Rupture: Pressure Cell Sonication Pressure Cell
Pretreatment: (a) (a) (b)
Solvent: Deionized Water Deionized Water Deionized Water
Soluble Nitroqen Release Data
(c) Number of Soluble Time Soluble Number of Soluble
Times Nitrogen of Nitrogen Times Nitrogen Through Release Sonication Release Through Release P. Cell (%) (Minutes) (%) P. Cell (%)
1 18.8 0 6.2 1 9.2
3 24.6 1 9.6 3 11.3
3 11.9
4 13.3
5 13.7
8 14.8 0'1 0'1
Table 4 (Continued). Thiobacillus thiooxidans Cell Rupture Experiments.
Type of Rupture:
Pretreatment:
Solvent:
Time of
Sonication (Minutes}
0
10
20
30
TEST IV Sonication
(b)
Deionized Water
TEST V Pressure Cell
(a)
pH 7.9 Buffer (d)
Soluble Nitrogen Release Data
Soluble Number of Nitrogen Times
Release Through (%} P. Cell
0.5 1
5.5 3
8.8
9.0
(a) Cells were washed three times with deionized water.
Soluble Nitrogen
Release (%)
--48.5
67.9
(b} Cells were treated with 2g of AG-1X2 and 2g of IR-120 ion-exchange resin. (c) The per cent soluble nitrogen is defined as: nitrogen in supernatant centrifuged
at 13,000xg for 10 minutes divided by total nitrogen in suspension before treatment. (d) Buffer prepared by mixing: 936 ml of M/15 Na 2Po4 and 64 ml of M/15 KH 2Po 4 .
0'\ ......
68
which were not available as standard solutions were taken
from data given by Smith (97). Table 5 gives the amino
acids found in hydrochloric acid digests of cell walls using
paper chromatography.
The sugars were separated by single dimensional descend
ing paper chromatography. They were identified by spraying
with silver nitrate spray reagent. The solvent used was
butanol, acetic acid, and water in the same proportions as
given in the preceeding paragraph. The sugars were identi
fied by comparing R values with those of known sugar g
samples. The sugars identified by paper chromatography in
the IR-120 ion exchange digestion mixture were: rhamnose,
glucose, and galactose. Traces of the amino sugar
glucosamine were found on the amino acid papergrams.
Amino Acid Analysis. The results of amino acid
analyses of cell walls prepared by hydrochloric acid
hydrolysis and trypsin digestion followed by hydrochloric
ac · d h bl 6 These analyses are ~ ydrolysis are given in Ta e •
given on a gram per lOOg of cell wall basis, and on a gram
per 100 g of dry hydrolyzate basis. Column three of Table 6
g . J.'n amJ.'no acids after digestion ~ves the per cent reduction
With
for
the
trypsin. The cell walls were tested
!est for Contamination. · of light in . the absorptJ.on
contamination by measurJ.ng d b Salton (95) and
ultraviolet region as suggeste Y typical curve for the
Keeler et al. t from a pressure
cell Walls and a sample of the supernatan
( 96) • Figure 20 gives a --
Cell rupture.
69
Table 5. The Qualitative Amino Acid Content of
Thiobacillus thiooxidans Cell Walls.
Amino Acid
Alanine
Arginine
Aspartic acid
Cysteic acid
Cystine
Diaminopimelic acid
Glutamic acid
Glycine
Histidine
Leucine/Isoleucine
Lysine
Methionine
Methionine sulphoxide
Phenylalanine
Proline
Serine
Threonine
Tyrosine
Valine
(a) + + large, intenselY colored + =
+ + = fairly large spot;
+ = small but definite spot;
- no spot found.
Relative Amounts {a)
+ + +
+
+ +
+
+
+ + +
+ +
+ +
+ +
+ +
+ +
+
+ +
+ +
+ +
+ +
spot on chromatogram;
Table 6. The Quantitative Amino Acid Content of Thiobacillus thiooxidans Cell Walls.
Cell Wall Basis Dry Hydrolyzate Basis Trypsin Tryps1n
Digestion Digestion Followed Loss of Followed Loss of
HCl by HCl Amino HCl by HCl ;..mino Digestion Digestion Acid Digestion Digestion Acid
Amino Acid (g/lOOg} (g/lOOg} ( % } (g/lOOg) (g/lOOg) ( %)
Alanine 2.20 0. 361 84.5 3.64 1.552 57.5 Arginine 1.77 0.020 98.9 2.94 0.086 97.0 Aspartic
Acid 2.72 0.395 85.4 4.52 1.696 62.4 Diamino-pimelic acid (a) (a) -- (a) (a) Glutamic acid 3.30 0.468 85.9 5.49 2.01 63.5 Glycine 1.45 0.221 84.9 2.41 0.948 60.6 Half-Cystine 0.05 TRACE -- 0.09 TRACE Histidine 0.58 0.054 91.5 0.97 0.230 75.3 Isoleucine 0.98 0.094 90.7 1.62 0.403 75.4 Leucine 2.40 0.321 86.6 3.99 1.380 65.4 Lysine 1.11 0.107 90.0 1.84 0.460 75.0 Methionine 0.53 TRACE 100.0 0.88 TRACE 100.0 Ornithine 0.16 0.087 43.7 0.26 0.374 -44.0 Phenylalanine 1.16 0.167 85.4 1.93 0.719 62.7 Proline 1.16 0.120 89.7 1. 9 3 0.517 73.1 Serine 1.37 0.301 78.0 2.28 1.292 43.5 Threonine 1.11 0.187 82.9 1.84 0.805 56.0 Tyrosine 1.00 0.147 85.0 1.67 0.632 62.3 Valine 1.59 0.254 84.3 2.63 1.092 58.6
24.64 3.304 86.6 40.93 1.320 67.9
(a) Its presence is suspected but was not quantitatively established. -....]
0
-dP -z 0 H 8 ~ ~ 0 tl.l
~
~ H 8 < ...:I IJ;:I ~
100
90
80
70
60
50
40
30
20
10
0 230
supernatant
cell walls
240 250 260 270 280 290 300 310 320 WAVE LENGTH (mf.l)
Figure 20. Ultraviolet Absorption of Cell Walls and Supernatant from Pressure Cell Rupture.
330
-J ........
72
IV. DISCUSSION
A. DISCUSSION OF RESULTS
Surfactant Studies. Investigations by Schaeffer (65)
and Jones and Benson (66) indicated that Thiobacillus
thiooxidans produced a large amount of surface active agent.
Schaeffer identified this agent as phosphatidyl inositol,
while Jones and Benson have reported that the agent is
phosphatidyl glycerol. The surface active agent presumably
aids in wetting the sulfur and thus aids in the oxidation of
the sulfur.
Earlier work by Starkey, Jones, and Frederick (62) with
surfactants indicated that of eight surfactants studied only
Tergitol 08 and Tween 80 accelerated the oxidation of sulfur
in shake flask studies.
The surfactants Tergitol 08 and TWeen 80 were studied
in this work for their ability to reduce the lag phase of
growth during the culture of T. thiooxidans. It was reasoned
that a reduction of the lag phase would decrease the time
required to produce a given amount of cells.
The 08 Was abandoned after visual obser-use of Tergitol
vations of its effect on the sulfur suspended in the medium.
Aeration of the shaken media containing Tergitol 08 for
h f in the media to rise 5 ort periods of time caused the sul ur
to the surface. The sulfur was retained there by a layer of
f · 1 08 tested were 50 to oam. The concentrations of Terg~to
sao ppm. It was assumed that if the sulfur floated on the
73
surface of the fermentation vessel ' then the organism would
not have ample access to the sulfur and the rate of sulfur
oxidation would be reduced. Foam also created operating
problems in the fermentor.
Visual observations of media containing Tween 80 indi
cated that this surfactant did not have the undesirable
properties of Tergitol 08. Tween 80 allowed immediate
dispersal of sulfur in shake flasks and foaming seemed to
be minimal.
Shake flask studies of growth using Tween 80 as a
Wetting agent were undertaken. The total amount of acid
produced by the organism was reduced by the presence of
Tween 80 in the medium. Tween 80 at a concentration of
12.5 ppm did, however, increase the initial rate of oxida-
tion (Figure 18, p. 62) This initial increase in rate was
probably the result of the ease with which the organisms
could attach themselves to the sulfur particles when a sur-
factant had been added to the medium. However, Tween 80
must have also inhibited the growth of T. thiooxidans and
consequently the oxidation of sulfur to some extent.
Pilot scale fermentations were carried out using 12.5
and 50 ppm concentrations of Tween 80. These tests (just
as did the shake flask studies) indicated that a concentra
tion of 12.5 ppm Tween 80 increased the initial rate of
growth. very little growth occurred at a concentration of
50 ppm Tween 80 (Figure 19, p. 65). In shake flasks growth
still occurred at 230 ppm Tween 80 (Figure 17, P· 61). The
growth in the fermentor was limited because the violent
aeration and agitation caused the medium to foam. The
sulfur was retained on the surface of this foam where it
was not accessible to the organism.
It was concluded that the use of Tween 80 or Ter
gitol 08 was not effective in increasing the yield of
T. thiooxidans cells in pilot scale fermentors.
74
Cell Rupture. Experiments were conducted to determine
the most efficient manner for rupturing the cells. The
amount of protein released from washed cells was evaluated as
an index of the degree of cell rupture. Initially attempts
were made to measure the protein released using the biuret
method (93) and the spectrophotometric method (94). Neither
of these methods was found applicable for the solutions
involved. The biuret method seemed to give consistent data,
although not all of the material precipitated with trich
loroacetic acid could be completely dissolved in some cases.
The amount of protein determined by the biuret method dif
fered greatly from that determined by the spectrophotometric
method. Because of the difficulties encountered with the
determination of protein, soluble nitrogen was also evaluated
as a means for determining the degree of cell rupture.
Nitrogen was determined by the Kjeldahl procedure. This was
found to be a reliable method and, consequently, it was used
for all the cell rupture studies.
suzuki and Werkman (57) found T. thiooxidans resistant
to rupture by sonication unless the cells were pretreated
75
with a mixture of anionic and cationic ion exchange resins.
They treated the cells with IR-120 and IR-4B ion exchange
resins in distilled water. A similar pretreatment using
AG-1X2 ion exchange resin to replace IR-4B was tried. This
pretreatment was not found to be a satisfactory method for
increasing the degree of cell rupture. In fact, it reduced
the amount of soluble nitrogen in the final suspension of
broken cells, when compared to that of untreated cells. Part
of this decrease might have been due to the fact that the
ion exchange resins removed some of the nitrogen initially
present in the samples. As shown in Table 4, Test II, p. 66
the initial amount of soluble nitrogen present in an
untreated sample was 6.2%, while in a pretreated sample
(Test IV) only 0.5% was present. The soluble nitrogen
initially present could have come from the rupture of cells
while in the frozen state, or as a result of insufficient
washing of the cells prior to treatment. The ion exchange
resins might have also affected the permeability of the cell
wall allowing some nitrogenous material to escape without
actual rupture of the cells.
Since a very small release of the total nitrogen took
place in deionized water, (Table 4, p. 66) a basic buffer
(pH 7.9) was tested as a medium for cell rupture. Because
T. thiooxidans can withstand acid conditions but cannot
live under basic conditions it was felt that the basic
buffer might aid in cell rupture. This basic buffer (pH 7.9)
was adopted for routine work, with no further study at other
hydrogen ion concentrations, because it gave a five or six
fold increase in soluble nitrogen release as compared to
deionized water (Table 4, p. 66).
The pressure cell rupture method was adopted for
routine work because it gave more soluble nitrogen than
sonication. Comparison of Test I with Test II, Table 4,
p. 66, indicates that putting the cells through the pres
sure chamber once gave more rupture than eight minutes of
sonication. Tests III and IV of Table 4 show that about
30 minutes of sonication gave about the same amount of
soluble nitrogen as did the running the cells through the
pressure cell once.
76
Hydrolysis Methods. Cell walls hydrolyzed for 2 hours
at 100-103 c with 2 N sulfuric acid gave no indication of
sugars or polyols when the solutions were chromatographed.
The reason sulfuric acid did not give sugar residues is
probably linked to the high resistance of the organisms to
sulfuric acid.
Keeler et al. (96) were able to solubilize the indi-
vidual monosaccharides in the cell walls of Vitrio fetus
by adding the ion exchange resin Permutit Q to the cell
walls and heating the suspension for 24 hours at 120 C in
a sealed tube. A similar digestion procedure using the ion
exchange resin IR-120 gave solutions in which rhamnose,
glucose, and galactose could be detected by paper chroma-
tography.
77
Rhamnose was easily detected by the chromatographic
method employed. A special procedure was used to separate
glucose and galactose since they have essentially the same
Rg values in the chromatographic solvent employed. The
glucose in the sample was destroyed using glucose oxidase.
The cell wall hydrolyzate, glucose, galactose, and a mix-
ture of glucose and galactose were incubated with glucose
oxidase at room temperature for six hours. The solutions
were filtered and evaporated under vacuum in a Rinco
evaporator until two or three drops remained. All the
samples containing glucose, including the cell wall hydro
lyzate, gave a spot on a chromatogram corresponding to
gluconic acid, the expected product from the action of
glucose oxidase on glucose. A galactose spot was observed
on the chromatograms containing this monosaccharide.
A The Sugars found in the cell nalysis of Cell Walls.
Walls hydrolyzed with IR-120 ion exchange resin were
rh Sugars found in other amrose, glucose, and galactose.
cell 1 271) arabinose, fucose, wa ls are (95, 29, p. : 'b and tyvelose.
galactose, glucose, mannose, rhamnose, r~ ose, on the amino acid
Traces of glucosamine were also detected
Papergrams of the cell wall hydrolyzates of T. thiooxidans.
· was not The . 'd or galactosam~ne presence of muram~c ac~
detected. Salton (29, P· 114) has mentioned that all been found in bacter
three of the above amino sugars have
f g lucosamine present The small amount 0 ial cell walls.
78
in the cell wall points to the similarity of the cell walls
of T. thiooxidans and cell walls of Gram-negative bacteria
Figure 2, p. 20).
The analysis of cell walls hydrolyzed with hydro-
chloric acid differed somewhat depending on the method of
analysis. The results of the analysis using the Amino
Acid Analyzer (Table 6, p. 70) indicated that three com
pounds not found by paper chromatography (Table 5, p. 69)
were present in the cell wall hydrolyzates. They were
ornithine, phenylalanine, and tyrosine. The Amino Acid
Analyzer did not detect the presence of diaminopimelic
acid, glucosamine, cysteic acid, and methionine sulphoxide.
The reason phenyalanine was not detected by paper chroma
tography was that its Rf value is very similar to that of
leucine and isoleucine and the spots may have overlapped
on the chromatograms. Likewise, tyrosine has an Rf value
very near that of valine and methionine. The reason
diaminopimelic acid did not show up in the Amino Acid
Analyzer was probably due to the proximity of its peak to
that of methionine and isoleucine in the amino acid analy
sis. Likewise, glucosamine and valine would probably form
only one peak. The small amounts of cysteic acid and
methionine found by paper chromatography might be due to
the variations of the composition of the cell walls.
A comparison of the more qualitative data from paper
chromatography, (Table s, p. 69) with data given by Salton
(95), Table 7, indicated that it is very difficult to
Amino Acid
Alanine Arginine Aspartic acid Cysteic acid Cystine Diaminopimelic
acid Glutamic acid Glycine Histidine Leucine Isoleucine Lysine Methionine Methionine sulphoxide Phenylalanine Proline Serine Threonine Tyrosine Valine
Table 7. The Qualitative Amino Acid Content of Some Gram-
positive and Gram-negative Bacterial Cell Walls (95).
Streptococcus pyogenes
+ + + (a) +
+ + +
+ + + + +
+ + + + +
+ + +
+ + + +
+ +
Gram-positive Organisms
Streptococcus faecalis
+ + +
+
+ + +
+ + + +
+ +
+
Micrococcus lysode1ktus
+ + +
+ + + +
+
---
Sarcina lutea
+ + +
+
+ + + + +
+ +
--
-
Bacillus subtillus
+ + +
+
+ + + + + +
+
+ +
+ +
+
-...)
~
Table 7 (Continued). The Qualitative Amino Acid Content of Some Gram-positive
and Gram-negative Bacterial Cell Walls (95).
Gram-negative Organisms
Amino Acid Escherichia
Alanine Arginine Aspartic acid Cysteic acid Cystine Diaminopimelic acid Glutamic acid Glycine Histidine Leucine/Isoleucine Lysine Methionine Methionine sulphoxide Phenylalanine Proline Serine Threonine Tyrosine Valine
(a)+ + + = large, intensely colored + + = fairly large spot
+ = small but definite spot no spot found
col1
+ + + (a) +
+ + + -+
+ + + + + -
+ + + + +
+ + + +
+ + + +
+ + +
spot on chromatogram
Salmonella pullorum
+ + + +
+ +
+ + + +
+ + +
+ + +
+ + + +
+ +
00 0
81
distinguish between Gram-positive and Gram-negative bacteria
on the basis of the amino acids present in the hydrolysis
of cell walls. One obvious disadvantage of this system is
that each person may assign the "+++", "++", "+", and "-"
different relative values. Comparison of the quantitative
amino acid data shown in Table 6, p. 70, with the data
reported by Salton (29, p. 254-255) and shown in Table 8,
shows however, that T. thiooxidans cell walls more closely
resemble those of Gram-negative than those of Gram-positive
organisms. It should be noted that for Lactobacillus casei
(a Gram-positive bacterium} the three major amino acids
make up 65% of the amino acids present, and the five major
components make up 80% of the amino acids present. In
Streptococcus faecalis (another Gram-positive organism)'
the three major amino acids make up 90% of the amino acids
present. For T. thiooxidans and the Gram-negative bacteria
no single amino acid makes up more than about lO to lS% of
the total amino acids present.
Of Of amino acids per Summation of the number grams
1 Gram-negative cell 00 g of cell wall indicates that the
f inc acids Walls are composed of a greater percentage 0 am
Therefore, the than Gram-positive cell walls (Table 8>·
d . T thiooxidans more closely amount of amino acids faun J.n ~ ~~~~---
't' bacteria. resembles that found in Gram-posJ. J.Ve
acJ.'ds found on hydrolysis is The amount of amino
· The data dependent upon the conditions of hydrolys~s.
Table B. The Quantitative Amino Acid Content of Some Gram-positive
and Gram-negative Bacterial Cell Walls (29, p. 254-255).
Gram Positive Organisms Gram Negative Organisms
Amino Acid
Alanine Arginine Aspartic acid Diaminopime1ic
acid Glutamic acid Glycine Half-Cystine Histidine Isoleucine Leucine Lysine Methonine Ornithine Phenylalanine Proline Serine Threonine Tyrosine Valine Total
Lactobacillus casei
g/lOOg {a)
8.4 - (b)
3.1
6.7 7.2 1.0
1.4 1.4 2.3
0.6 1.1
1.4 34.6
Streptococcus faecalis
g/lOOg
12.0
0.8
5.4 0.2
0.4 0.4 4.5
0.2 0.2
0.24 24.3
Escherichia coli
g/lOOg
5.6 3.8 7.1
+ (b) 6.9 3.1
0.9 3.7 5.3 4.0 0.7
3.0 1.5 3.7 3.8 3.3 3.4
59.8
(a) All values are given on a cell wall basis. (b) ~-" indicates that the concentration was not estimated;
"+" indicates that the amino acid was present.
Pseudomonas aeruginosa
g/lOOg
5.1 1.3 9.3
3.2 5.8 7.1
2.8
1.7
7.3
5.4
4.2 3.9
57.1
Salonella bethesda
g/lOOg
4.3 0.4
14.5
2.1 4.9 5.5
1.6
0.5
4.1
2.6
2.5 2.0
45.0
(X)
N
given in Table 8 are for the hydrolysis of the cell walls
in 6 N hydrochloric acid at a temperature of 100 to 105 c '
83
for periods from 12 to 24 hours. Although this is the most
general method of hydrolysis, Salton (29, p. 105) has
pointed out that the release of amino acids may be maximal
for a shorter digestion period. Salton also pointed out
that the maximum amount of diaminopimelic acid and lysine
was formed after only about 8 hours of hydrolysis in 4 or
6 N hydrochloric acid at 105 C. It is possible that if
different conditions of digestion were employed for the
hydrolysis of T. thiooxidans cell walls the amount of amino
acid per 100 g of cell wall might have been brought into
better agreement with those of Gram-negative organisms. If
cell walls from various microorganisms were digested under
similar conditions but for varying periods of time, such as
2 hour intervals over the range of 2 to 24 hours, one could
then determine the optimum digestion period. This diges
tion period would probably be different for each organism
examined.
Comparison of the molar ratios of amino acids present
in cell walls, Table 9, also indicates that the cell wall of
T. thiooxidans is more like that of Gram-negative than Gram
positive bacteria. The molar ratios of glycine and serine
are more like those of the Gram-negative than the Gram
positive organisms. The molar ratios of amino acids in
~ thiooxidans are more like those of the Gram-negative
Table 9. Molar Ratios of Some Amino Acids in Cell Walls.
Organism Lysine Glutamic Acid Glycine Serine Alanine
Escherichia coli 1.0 1.7 1.5 1.3 2.2
Pseudomonas aerug~nosa 1.0 3.4 8.3 4.4 4.9
Salmonella bethesda 1.0 9.7 21.0 7.2 14.1
Thiobacillus th~oox~dans 1.0 2.9 2.6 1.7 3.3 -Lactobacillus casei 1.0 3.1 0.9 0.4 6.0
StreEtococcus ·faecalis 1.0 1.2 0.1 0.1 4.4
(a} Data taken from Tables 6 and 8, p. 70 and 82.
(a)
Gram-Reaction
+
+
+
ro ~
85
bacterium Escherichia coli than any of the other bacteria
listed in Table 9.
The amino acid analysis of the trypsin digested cell
walls indicated that 87% of the cell wall amino acids were
removed. The per cent reduction in amino acids was calcu-
lated as follows:
grams of amino acid per 100 g of cell wall in the hydrochloric acid digest
grams of amino acid per 100 g of cell wall in the trypsin digest
grams of amino acid per 100 g of cell wall in the hydrochloric acid digest
X 100
using data obtained from Table 6, p. 70. If it is assumed
that trypsin digestion removed all the amino acids except
those in the glycosaminopeptide, and that the percentage of
amino acids in the cell wall and the glycosaminopeptide were
constant, then 13% of the cell wall could be considered to
be glycosaminopeptide. This is well within 10 to 20 % range
cited by Salton (29, p. 99-100) for glycosaminopeptide in
Gram-negative cell walls. It is unusual to find ornithine as a component of cell
1 . has not been found in
wa ls. To the author's knowledge 1t found may have been an
any other cell walls. The ornithine
artifact formed during the cell rupture procedure. orni-
thine ;s product of arginine formed under ~ a degradation
basic conditions. peptide bonds where The enzyme trypsin is specific for
. ·ne or L-lysine. the d by L-arg1n1
carboxyl group is donate
The enzymes will not hydrolyze peptides composed entirely
of ornithine (98).
Trypsin digestion removed 99% of the arginine, 100%
of the methionine, and only 44% of the ornithine present
~n the cell walls of T. thiooxidans. From these data the
sequence of the peptide in which arginine and ornithine
were located cannot be ascertained, but most of the
arginine residues must have been located near lysine ~n
order to be completely removed. Ornithine, on the other
hand, must have been located in peptides which contained
fewer lysine and arginine bonds. If ornithine were formed
from arginine during the rupture procedure, it would most
likely have been formed at the surface of the cell wall.
The trypsin digestion data, however, would indicate that
arginine in the surface layer was a portion of a peptide
with a very small amount of lysine, and this could account
for the small loss of ornithine formed from the arginine.
Another possibility is that the glycosaminopeptide which
86
is resistant to trypsin digestion, contained a high propor-
tion of ornithine.
In order to determine if ornithine is initially
present in the cell walls enough cells must be ruptured in
deionized water to make amino acid analysis possible. If
ornithine is not found in this analysis this would indicate
that the ornithine was formed during the cell rupture at
pH 7.9.
87
From the above results it may be concluded that the
cell walls of T. thiooxidans more closely resemble the cell
walls of Gram-negative than the Gram-positive bacteria and
that there is a possibility that the cell walls of
T. thiooxidans contain ornithine.
Test for Contamination. Cell walls were tested for
contamination by intracellular material (nucleic acid) by
measuring their absorption of light in the ultraviolet
range. This procedure measured the contamination by intra
cellular material, and was suggested by Salton (29, p. 42-63)
and Keeler et al. (96). The results, shown in Figure 20,
indicate that the cell walls did not absorb at 260 m~ as did
the supernatant from the cell rupture. This indicates that
the cell walls were not contaminated with intracellular
material. Salton (29, p. 96) has stated that the high light
scattering of the cell walls might mask the absorption at
260 m~ resulting from the presence of nucleic acid.
88
B. LIMITATIONS
Salton (29, p. 42-63} reported that the most useful
guide for the determination of cell wall homogeneity was the
electron microscopic examination. With the aid of electron
microscopy one could determine if the cell walls were con-
taminated with flagella. The flagella are very easily
removed from bacteria (96). Keeler et al. (96) used dif
ferential centrifugation at 68,000 x g for 1 hour to harvest
flagella of Vibrio fetus from other cell fragments. The
cell walls of Thiobacillus thiooxidans in the present study
were harvested at 8,400 x g for 20 minutes. Therefore,
the flagella were probably lost during the differential
centrifugation. Two criteria of homogeneity were also
mentioned by Salton (29, p. 42-63}: spectroscopic examina
tion of cell walls for intracellular chemical components,
and the separation of cell walls in sucrose density gradients.
At the time this experiment was conducted, an electron
microscope was not available on this campus. For this
reason electron micrographs could not be used as an indica
tion of homogeneity. The cell walls were, however, collected
using the sucrose densitY gradient method. The examination
of the ultraviolet absorption of the cell walls gave no
evidence of the presence of intracellular components.
Therefore, the author felt justified in assuming that the
cell walls were homogeneous in nature.
89
The ATCC strain number 8085 of !:._ thiooxidans was used
in this experiment. Other strains of !.:_ thiooxidans cell
walls might exhibit differences in cell wall composition.
V. CONCLUSIONS
From the results obtained in this investigation the
following conclusions were made:
90
(1) The use of the surfactants Tergitol 08 and Tween 80 was
not effective in increasing the cell yield of
Thiobacillus thiooxidans in pilot scale fermentors.
(2) The best rupture of T. thiooxidans cells was obtained
using a basic buffer (pH 7.9) and the pressure cell
rupture technique.
(3) The hydrolysis of cell walls for sugar residue was best
performed using digestion with the ion exchange resin
IR-120 and not sulfuric acid.
(4) The cell wall of T. thiooxidans was found to have an
amino acid content more like that of Gram-negative than
Gram-positive organisms.
(5) The possibility that ornithine was present in the cell
walls of T. thiooxidans was indicated.
(6) The cell wall of T. thiooxidans was found to contain the
sugars, rhamnose, glucose, and galactose. It also con
tained small amounts of the amino sugar glucosamine.
91
VI. RECOMMENDATIONS
The following method is suggested for large scale
growth of the organism Thiobacillus thiooxidans. It depends
on a semi-batch type fermentation. The fermentor should be
charged with medium and inoculated with organisms. After
the organisms have reached maximum growth, 80 to 90% of the
medium should be withdrawn and replaced with fresh medium.
The cycle should then be repeated.
Recommendations for further study include:
(1) Electron microscopic examination of cell walls for
homogeneity and surface appendages.
(2) Rupture of enough cells in deonized water to determine
if ornithine is present in walls not ruptured under basic
conditions.
(3) Study to determine if trypsin is specific for ornithine
residues found in peptides.
(4) Study to determine if trypsin will liberate amino acids
from glycosaminopeptides.
(5) Further study of cell wall hydrolysis methods.
(6) Examination of the cell walls for the presence of lipids.
(7) Quantitative study of the cell wall monosaccharide
composition.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
92
VII. B I BLI OG E.APHY
Braley, S ·A· , "Coal Mine Effl '-..lents " Presented at the Symposium on Fossil Fuel:::; and 1 Environmental Poll uti on of the Di visior1 of Fuel Chemistry 15lst National Meeting of the ~erican Chemical S~ciety, Pittsburgh, Pennsylvania (March 23, 1966).
Temple, K. L. and Colmer, A. R. , "The Formation of Acid Mine Drainage, " Mining E::a1.gineering, ir 1091-1092 (1951).
Carpenter, L. V. and Herndon :::::L.K., "Acid Mine Drainage , . . from Bituminous Coal Min-es," West Virg1.n1.a University Engineering s-tation Bulletin, No. 10, 19 (1933).
Colmer, A. R. and Hinkle, M.E. , "The Role of Microorganisms in Ac .:i.d Mine D .::rainage," Science 1 ~, 253-256 {1947) •
Colmer, A. R., Temple , K. L. an .d Hinkl~, M.E., ''An Iron-h A d Drainage of Some Oxidizing Bacte .:r i urn from. t e cl. 59 , 317_32s (1950).
Bituminous Coal Mines," -.J. Bact.,
Harmsen, G.W., Quispel, A. an and Bacteriological Oxid Plant and Soil, ~, 43-45
Hinkle, M.E. and Koehler, W.A.. Microorganisms in Acid K No. 2301, Coal Tech., Ala 139-148 {1948) •
d Otzen, D., "The Chemic~l ation of Pyrite in soil,
{1952).
"The Action of certain i~e Drainage, n Tech. Pub. • Inst. Min. Met. Eng.,
"Bacterial ActivitY on Leathen, W.W. and BJ?aley, s.z- ·~ciated with Coal," Bact.
Sulfuric ConstL tuents A 2 ~- 2 2 (1950). Pro c • , Abs t. of Paper'
"The Effects of Iron Leathen, w.w. and Bra~ey, s.A- 'tain constituents of
Oxidizing Bacter3..a on ce :c Abst. of Paper' Bituminous CoaL,.. Bact. proc.,
21-22 (1951). . d Mcintyre 1 LOis 0 · '
Leathen, w.w., BraleY, S~A·. a.:n the Formation of ~cld "The 1 f BacterJ.a l.n tituents Assoclated Ro e o •t'c cons 1 61-64 from Certain Su1furl. ! ,A.._ppl. Microbiol' -' with Bituminous coal' (1953).
Leathen, w.w. and Ferrous Iron Water," Soc· Paper, 64-65
"The Oxidati~n of d .;son I<- ..14·' d 1·n Acid M1ne
Ma. ....... ' · Foun f bY Bacterl.a r ioloqists' Abst. o of Am. Bact e (1949).
93
12. Leathen, W.W., M<:=Intyre, ~ois D. and Braley, s.A., "A Study c;>f A<:=~d ~ormat~on by Iron Oxidizing Bacteria Found ~n B~turn~nous Coal Mine Drainage," Bact. Proc., Abst. of Papers, 15-16 (1952).
13. Marchlewi tz, B. and Schwartz, W. , 11 Microbe Association of Acid Mine Water," Z. Allgem. Mikrobiol., 1, 100-144 (1961). -
14. Temple, K. L. and Colmer, A. R., "The Formation of Acid Mine Drainage," Proc. 6th Ind. Waste Conf., Purdue Univ. Eng. Bull. Extension Ser. No. 76, 285-291 (1951).
15 • Temple, K. L. and Del champs, E. W. , 11 Autotrophic Bacteria and the Formation of Acid in Bituminous Coal Mines, 11 Appl. Microbial., _!, 255-258 (1953) ·
16. Temple, K.L. and Koehler, W.A., "Drainage from Bituminous Coal Mines," W.Va. Univ. Bull.' Eng. Expt. Sta. Research Bull. No. 25, 1-35 (1954).
17. Zarbina, z .M., Lyalikova, N.N. and ~hrnu~, E. I.' 11 Investigation of Microbial Ox~dat~on of Coal Pyrite, II Izvest. Akad. Nauk. s.s.s.R., Otdel, Tech. Nauk., Met. i Toplino, No. 1, 117-119 (1959) ·
18 • Hodge, W. W. , "Effect of Coal Mine Drainage on West. Virginia Rivers and Water Supplies,,. W.Va. UnlV.
19.
20.
21.
22.
23.
E Res. Bull. 18, 30 (1938) · ng. Expt. Sta.
. . 1 Report to the R~ver Pollution Survey' F~na .. 11 Acid Mine River Corruni ttee, Supplement C '. t A ency Drainage Studies. Federal Secur~ Y g ' Public Health Service (1942).
Ohio Ohio
u.s.
Mining Congr. J., United States War Department Report,
12, 523-524 (1926). . "Design and Economics
G~rard, Lucien and Kaplan' R.A.' t Plant--of an Acid Mine Drainage Trea~e~t the !51st Operation Yellowboy," Presen~e an chemical society National Meeting of the ~er~~arch 24, 1966 · at Pittsburgh, Pennsylvan~a,
Disposal "Subsurface
Linden, K. V. and Stefanko, ~obert, nted at the 151S~ . of Acid Mine Drainage' Pres~ an chemical soc let} National Meeting of the ~er~~arch 24, 1966 • at Pittsburgh, Pennsylvan~a,
Progr., ~, 11 " sci.
King I H. K. , "The Bacterial cell wa , 299-310 (1961).
24. Armstrong, J.J., Baddiley, J., Buchanan, J.G. and Carss, B., "Nucleotides and the Bacterial Cell Wall," Nature, 181, 1692-1693 (1958).
94
25. Martin, H. H., "Biochemistry of Bacterial Cell Walls," Annual Rev. of Biochemistry, 35 Part II 457-484 (1966). _, ,
26. Work, Elizabeth, "Biochemistry of Bacterial Cell Wall," Nature, 179, 841-847 (1957).
27. Zilliken, Friedrich, "Chemistry of Bacterial Cell Walls," Federation Proc., 18, 966-973 (1959).
28. Mandelstam, J. and Rodgers, H.J., "The Incorporation of Amino Acids into the Cell Wall Mucopeptide of Staphylococci and the Effect of Antibiotics on the Process," Biochem. J., 72, 654-662 (1959).
29. Salton, M.R.J., The Bacterial Cell Wall, (1964), Elsevier Publishing Co., New York.
30. Breed, R.S., Murry, E.G.O. and Hitchens, A.P., Bergey's Manual of Determinative Bacteriology, 6th ed., (1948}, Williams and Wilkins Co., Baltimore, p. 79.
31. Waksman, S.A. and Joffe, J.S., ~The Oxidation of Sulfur by Microorganisms," Proc. Soc. Exptl. Biol. Med., 18, 1-3 (1920).
32. Waksman, S.A. and Starkey, R.L., "The Growth and Respiration of Sulfur--Oxidizing Bacteria," J. Gen. Physiol., ~, 285-310 (1923).
33. Waksman, S.A. and Joffe, J.S., "Microorganisms Concerned in the Oxidation of Sulfur in Soil II. Thiobacillus thiooxidans, a New Sulfur-Oxidizing Organlsm Isolated from Soil," J. Bact., z, 239-256 (1922).
34. Starkey, R.L., "Isolation of Some Bacteria which Oxidize Thiosulfate," Soil Sci., i2_, 197-218 (1935).
35. Umbreit, w.w., Vogel, H.R. and Vogler, K.G., "The Significance of Fat in Sulfur Oxidation by Thiobacillus thiooxidans," J. Bact., 43, 141-148 (1942).
36. Baalsrud, Kjell, "Some Aspects of Physiology of Thiobacilli," Autotrophic Micro-organisms, 4th Symposium of the Soc. for General Microbiology, London, (1954), Cambridge University Press, p. 54-67.
95
37. Starkey~ R.L:, "The Carbon and Nitrogen Nutrition of Th7o~a~1llus thiooxidans, an Autotrophic Bacterium Oxldlzlng Sulfur Under Acid Conditions " J B t 10, 165-195 (1925). ' · ac ·'
38. Butler' ~· G · and Umbrei t, W. W. , "Absorption and Uti1i
za~1on ~f Organ~c M~tter by the Strict Autotroph, Th1obac1llus th1oox1dans, with Special Reference to Aspat1c Ac1d," J. Bact., 91, 661-666 (1966).
39. O'Kane, D.J., "The Presence of Growth Factors in the Cells of the Autotrophic Sulfur Bacteria," J. Bact., 43, 7 (1942).
40. Starkey, R.L., "The Physiology of Thiobaci11us thiooxidans, an Autotrophic Bacter1um Ox1dizing Sulfur Under Acid Conditions," J. Bact., 10, 135-
41.
42.
43.
44.
45.
46.
4 7.
48.
163 (1925). -
Parker, C.D. and Prisk, Joyce, "Oxidation of Inorganic Compounds of Sulfur by Various Sulfur Bacteria," J. gen. Microbial.,~, 344-364 (1953).
Suzuki, Isamu and Werkman, C.H., "Glutathione and Sulfur Oxidation by Thiobacillus thiooxidans," Nat. Acad. of Sci. Proc., 45, 239-244 (1957).
Waksman, S.A., "Microorganisms Concerned in the Oxidation of Sulfur in the Soil III. Medium Used for Isolation of Sulfur Bacteria from Soil," Soil Sci., 13, 329-336 (1922).
Waksman, S.A. and Joffe, J.S., "The Oxidation of Sulfur by Microorganisms to Sulfuric Acid and Transformation of Insoluble Phosphates into Soluble Forms," J. Biol. Chern.,~' 35-45 (1922).
Suzuki, Isamu and Werkman, C.H., "Phosphoenolpyruvate Carboxylase in Extracts of Thiobacillus thiooxidans, a Chemosynthetic Bacterium." Arch. Biochem. Biophys., 72, 514-515 (1957).
Vogler, K.G., "The Presence of an Endogenous Respir~tion in the Autotrophic Bacteria," J. Gen Phys1ol., 25, 617-622 (1942}.
Vogler, K.G., "Studies on the Metabolism of Autot~ophic Bacteria II. The Nature of the Chemosynthet1c Reaction," J. Gen. Physiol., 26, 103-117 (1942).
Vogler, K.G. and Umbreit, W.W., "Studies on the Metabolism of Autotrophic Bacteria III. ~he . Nature of the Energy Storage Material Act~ve 1n the chemosynthetic Process," J. Gen. Phys1ol., 26, 157-167 (1942).
I I I I I I I I I I I
I I I I I I I I I I I I I I I
I I I I
~ I
I I I I I
I I I
I I I I I I I
I I I I I I I
I I
I
I I I I I I I I I I I
49.
so.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
96
LePage, G.A., "The Bi. h · The Metabolism 0~c T~~~~trr 1~f Autotrophic Bacteria. Absence of Oxid · ~0 ac1. us thiooxidans in the 255-262 (1942). -l..zable Sulfur," Arch. Biochern., _!_,
LePage, G.A. and Umb.:reit w w 11 h hydrate Esters . ' .• , .P osphorylated Carbo-
~n Autotroph~c Bacteria," J. Biol. Chern., 14 7, 2631-271 c1943).
LePage, G.A. and Uml:::):reit w w "The Occ f . , , • ., urrence o Adenos7ne"- 3 - Triphosphate in Autotrophic Bacter1.a, J. Biol. Chern., 148, 255-260 (1943).
Barker, H. A. a~d Kornberg, Arthur, "The Structure of th7 Ad7nos~n;triphosphate (ATP) of Thiobacil1us th~oox~dans, J. Bact., 68, 655-661 (1954).
Baalsrud, Kjell and Baalsrud, K.S., "The Role Phosphate in Carbon Dioxide Assimilation Thiobaci1li," Phosphorous Metabolism 2
h k • I I Jo n Hop ~ns Press, Baltimore, p. 544.-
of of (1952),
Umbreit, W.W., "Phosphorylation and Carbon Dioxide Fixation in Autotrophic Bacterium, Thiobacillus thiooxidans," J. Bact., 67, 387-393 (1954).
Newburgh, R. W., "Phosphorylation and Chemosynthesis by Thiobaci11us thiooxidans," J. Bact., 68, 93-97 (1954). --
Suzuki, Isamu and Werkman, C.H., "Chemoautotrophic Fixation of Carbon Dioxide by Thiobacillus thiooxidans," Iowa State Coll. J. Sc1.. , 32, 4 75-4 8 3 ( 1953) • --
Suzuki, Isamu and Werkman, C.H., "Chemoautotrophic Carbon Dioxide Fixation by Thiobaci11us thiooxidans . I. Formation of Oxalacetic Acid, " Arch. B~ochem. Biophys., 76, 103-111 (1958).
suzuki Isamu and Werkman, C.H., "Chemoautotropic C~rbon Dioxide Fixation by Thiobacillus thiooxidans II. Formation of PhosphoglycerJ.c AcJ.d,n Arch. Biochem. Biophys., 77, 112-123 (1958).
suzuki, Isamu and Werkman, C.H., "Glutathione Reductase of Thiobacillus thiooxidans," Biochern. J., 74, 359-362 ( 1960 J •
Asnis, R.E., "A Glutathione Reductase from Escherichia coli, 11 J. Bio1. Chern., 213, 77 (1955).
97
61. Knaysi, Georges, "A Cytological and Microchemical Study of Thiobacillus thiooxidans," J. Bact. 46 451-461 (1943}. ' _,
62. Starkey, R.L., Jones, G.E. and Frederick, L.R., "Effects of Medium Agitation and Wetting Agents on Oxidation o~ Sul~ur by Thiobacillus thiooxidans," J. gen. Ml.CrObl.ol., 15, 329-334 (1956). .
63. Jones, G.E. and Starkey, R.L., "Surface Active Substances Produced by Thiobacillus thiooxidans," J. Bact., ~, 788-789 (1961).
64. Vogler, K.G. and Umbreit, W.W., "The Necessity for Direct Contact in Sulfur Oxidation by Thiobacillus thiooxidans," Soil Sci., 51, 331-337 (1941).
65. Schaeffer, W.I., "Biochemical Studies with the Sulfur Oxidizing Bacterium Thiobacillus thiooxidans," Ph.D. Thesis (1964) p. 1-30, Rutgers Un1versity, New Brunswick, New Jersey. ·
66. Jones, G.E. and Benson, A.A., "Phosphatidylglycerol in Thiobacillus thiooxidans," J. Bact., 89, 260-261 (1965}. --
67. Vogler, K.G., LePage, G. A. and Urnbreit, W.W., "Studies on the Metabolism of Autotrophic Bacteria I. The Respiration of Thiobacillus thiooxidans on Sulfur," J. Gen. Physiol., 26, 89-102 (1942).
68. Fischer, D.J., Herner, A.E., Landes, Alma, Batlin, Alex, and Barger, J.W., "Electrochemical Observations in Microbiological Processes. Growth of Thiobacillus thiooxidans. I," Biotech. Bioengr., 7, 471-490 {1965). -
69. Fischer, D.J., Landes, Alma, Sanford, M.T., Herner, A.E. and Wiegand, C.J.W., "Electrochemical Observations in Microbiological Processes. Growth of Thiobacillus thiooxidans. II," Biotech. Bioengr., 71 491-506 (1965).
70. Salton, M.R.J. and Horne, R.W., "Studies of the Bacterial Cell Wall II. Methods of Preparation and Some Properties of Cell Walls," Biochirn. et Biophys. Acta, 7, 177-197 (1951).
71. Salton M.R.J., "The Nature of the Cell Walls of Some G;am-Positive and Gram-Negative Bacteria," Biochim. et Biophys. Acta, ~, 334-335 (1952).
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
98
Baddiley, J., Buchanan, J.G. and Carss B. "The Presen~e ?f R~bitol Phosphate in Bact~rial Cell Walls, Blochlm. et Biophys. Acta, 27, :220 (1958).
Janczur~, E., Perkins, H.R. and Rogers, "Teichuronic Acld a Mucopolysaccharide Present in Wall Preparations from Vegetative Cells of Bacillus subtilis,n Biochem. J., 80, 82-93 (1961).
Salton, M.R.J., "The Anatomy of the Bacterial Surface, 11
Bacterial. Revs., 25, 77-99 (1961}.
Fruton, J.S. and Simmonds, Sofia, General Biochemistry, 2nd ed., (1958), John Wiley and Sons, Inc., New York, p. 80.
Work, Elizabeth, "The Isolation of a-s Diaminopimelic Acid from Corynebacterium diphteriae and Mycobacterium tuberculosis," Biochem. J., 49, 17-23 (1951}.
Strange, R.E., Biochem. J., 64, 23P (1956); cited by Work, Elizabeth, "Biochemistry of the Bacterial Cell Wall," Nature, 179, 841-847 (1957).
Strange, R.E. and Kent, L.H., "Isolation, Characterization, and Chemical Synthesis of Muramic Acid," Biochern. J., 71, 333-339 (1959}.
Cummins, c.s. and Harris, H., 11 The Chemical Composition of the Cell Wall in Some Gram-positive Bacteria and Its Possible Value as a Taxonomic Character," J. gen. Microbial., 14, 583-600 (1956).
Salton, M.R.J., "Studies of the Bacterial Cell Wall. v. The Action of Lysozyme on Cell Walls of Some Lysozyme Sensitive Bacteria," Biochim. et Biophys. Acta, 22, 495-506 (1956).
Salton, M.R.J., "Improved Method for the Detection of N-Acetylaminosugars on Paper Chromatograms," Biochim. et Biophys. Acta, 34, 308-312 (1959).
Ghuysen, J.M. and Salton, M.R.J., "Acetylh~xosarnine Compounds Enzymically Released fro~ Mlcrococcus . lysodeikticus Cell Walls I. Isolatlon and ~omposltion of Acetylhexosamine and Acetylhexosamlnepeptide Complexes," Biochim. et Biophys. Acta, 40, 462-472 (1960).
99
83. Salton, M.R.J. and Ghuysen, J.M., "Structure of Di- and Tetrasaccharides Released from Cell Walls by Lysozyme and Streptomyces F. Enzyrne and the B (1~4)-N-~ce~ylh~xosaminidase Activity of these Enzymes, B1och1m. et Biophys Acta 36 552-554 (1959}. • , _,
84. Park, J.T., "Uridine-5'-pyrophosphate derivatives. I. Isolation from Staphylococcus aureus, II. A Structure Common to Three Derivatives III. Amino Acid Containing Derivatives " J. Biol: Chern, 194, 877-904 (1952). , -
85. Ghuysen, J .M., "Structure of the Disacch.aridepeptide Complexes Freed by B(l~4) N-acetylhexosarninidases from Micrococcus lysodeikticus Cell Walls," Bioch1m. et B1ophys. Acta, 47, 561-568 (1961).
86. Salton, M.R.J., "Bacterial Cell Wall. VIII. Reaction of Walls with Hydrazine and Fluorodini trobenzene 1"
Biochim. et Biophys. Acta, 52, 329-341 (1961).
87.
88.
89.
90.
91.
92.
93.
Primosigh, J. , Pelzer, H. 1 Maass, D. and Weidel, W. 1
"Chemical Characterization of Mucopeptides Released from the Escherichia coli B Cell Wall by Enzymic Action," Bioch1m. et BJ.ophys. Acta, 4 6, 68-80 (1961).
Pelzer, H. , "Chemical Structure of Two Mucopeptides Released from Escherichia coli B Cell Walls by Lysozyme," Biochem. et B1ophys. Acta, 63, 229-234 (1962).
Mandelstam, M.H. and Strominger, J.L., "On the Structure of the Cell Walls of Staphylococcus aureus (Copenhagen) , " Biochem. Biophys. Research Communs · 1
~, 466-471 (1961).
Ribi, Edgar and Hoyer, B. H. , "Purificati<;>n of Q. Fev;r Rickettsiae by Density Gradient SedJ.rnentatJ.onl J Immunol., 85, 314-318 (1960). . -
crum, E.H. I "The Submerged Culture of Thioba~illus thiooxidans in a Pilot Scale Ferrnentor, M.S. Thesis (1964) p. 21-27, Missouri School of Mines and Metallurgy, Rolla, Missouri.
T H "The Production of Glutamic Acid by Fermenta-t. · .n, " M S Thesis (1965) p 40-43 1 Missouri School 10 , • • • . . of Mines and Metallurgy, Rolla, MLssourJ..
Li,
· M "Deterrni-Gornall, A.G. 1 Bardawell, C.J. and Dav1d1 M. "'. t nation of Serum Proteins by Means of the B~~~f Reaction," J. Biol. Chern., 177, 751-766 (1 ·
100
94. Seaman, G.R., "Microbial Physiology and Biochemistry," (1963), Burgess Publishing Co., Minneapolis, p. 52-53.
95. Salton, M.R.J., "Studies of the Bacterial Cell Wall. IV. The Composition of the Cell Walls of Some Gram- Negative Bacteria," Biochim. et Biophys. Acta, 10, 512-523 (1953).
96. Keeler, R.F., Ritchie, A.E., Bryner, J.H., and Elmore, Jane, "The Preparation and Characterization of Cell Walls and the Preparation of Flagella of Vitrio fetus," J. gen. Microbial., 43, 439-454 (1966).
97. Smith, Ivor, Chromatographic and Electrophoretic Techniques, Volume I. Chromatography, (1960), Interscience Publishing Inc., New York, p. 89-94.
98. Dixon, Malcolm, and Webb, E.C., Enzymes, (1958), Academic Press Inc., Publishers, New York, p. 264.
101
VIII. ACKNOWLEDGEMENT
The author wishes to express his appreciation for the
patient guidance and counsel afforded by his advisor, Dr. D.
J. Siehr, throughout the course of this investigation.
Special thanks is expressed to the author's second
reader, Dr. s. G. Grigoropoulos, and to his typists, Miss
Janet Davidson and Mrs. K. W. Davidson, who spent many hours
rushing this text to completion.
102
IX. VITA
Edward H. Crum was born on September 21, 1940, at
South Charleston, v-lest Virginia where he obtained his
primary educRtion. He received his secondary education at
Saint Albans, West Virginia, graduating from high school in
June 1958.
In September of this same year he entered West Virginia
Institute of Technology where he received a B.S. degree in
Chemical Engineering in June 1962.
He was awarded a National Defense Act Fellowship in the
spring of 1962. This fellowship was awarded in the field of
Engineering Biochemistry.
He entered the. graduate school of The University of
Missouri School of Mines and Metallurgy in September 1962
where he received a M.S. in Chemical Engineering in Hay, 1964.
The title of his thesis was "The Submerged Culture of
Thiobacillus thiooxidans in a Pilot Scale Permenter."
He is a member of the American Institute of Chemical
E · Amer~can Chern~cal Society, and the Instrt~ent ng l.neers, ..... ......
Society of America.
He was married to Kathryn Ann Duckworth on November 29,
1963. They have one child William H. who was born October
28, 1964.