Table Of ContentTHE PENNSYLVANIA STATE COLLEGE
THE GRADUATE SCHOOL
DEPARTMENT OF BACTERIOLOGY
THE RESPIRATORY BEHAVIOR OF PSEUDOMONAS AERUGINOSA
ON GLUCOSE AND HYDROCARBONS
A Thesis
by
ROSLYN L. SCHUMAN
Submitted in partial fulfillment
of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
October 1943
APPROVED:
a
Head of Department of Bacteriology
Professor of Bacteriology
TABLE OF CONTENTS
page
I. INTRODUCTION............... 1
II. REVIEW OF THE LITERATURE ..... g
III. GLUCOSE AND HYDROCARBON RESPIRATION IN THE WAR
BURG RESPIROMETER
A. Experimental Procedure...................... 6
B. The Oxidation of Glucose
1. Results ......... 11
2. Discussion. ..... 17
0. The Oxidation of Cetane
1. Results...... 19
2, Discussion ......... 25
D. The Oxidation of Naphthalene
1. R e s u l t s 26
2. Discussion............................ 35
IV. THE RESPIRATORY MECHANISM OF GLUCOSE AND HYDRO
CARBON OXIDATION
A. Warburg Respirometer Studies
1. Experimental Procedure............... 37
2. Results
a. Effect of Pyocyanine on Glucose,
Cetane and Naphthalene Oxida
tion. ........................... 40
b, Effect of NaF, Iodoacetic Acid,
NaCN, NaNg and Urethan on Gluc
ose, Cetane and Naphthalene
Oxidation....................... 45
page
B, Spectroscopic Measurements of Cells of
Pseudomonas Aeruginosa
1. Experimental Procedure*.......... 54
2. Results
a. Absorption Spectra Studies 56
b. Effect of Respiratory In
hibitors on the Cytochrome
S y s t e m , 59
C, Xh-Senssron* 62
V, SUMMARY.,. 65
VI,
VII, ACKK 0\VL EDGMEN TS „ *«<re«asc'«4««*eo9ac4*s4<»aece«s£«'«<s 70
I. INTRODUCTION
In the past few years, the utilization of hydrocarbons by micro
organisms has been a subject of much investigation in these laborator
ies. It was shown by Stone, White and Fenske (1940) that certain gram
negative rods could attack crude oil and some of Its fractions. Straw-
Inski and Stone (1940, 1942), investigating the effect of eighteen
specific hydrocarbons, found that organisms apparently identical with
Pseudomonas aeruginosa could readily attack cetane, naphthalene and bi
phenyl. The chemistry of the dissimilation of these compounds was stud
ied by Strawinski and Stone (1942, 1945) and by Jezl (1942).
The present investigation of the respiratory behavior of Pseudo
monas aeruginosa on glucose and hydrocarbons was undertaken with a
threefold purpose. First, it was hoped that Information could be
obtained on the type of respiratory enzyme system which enabled Pseudo
monas aeruginosa to attack substrates such as hydrocarbons. Secondly,
it was of interest to compai'e this mechanism with that which functions
in the oxidation of a more common carbon source such as glucose. Lastly,
it was felt that the knowledge provided by this study could also supple
ment the chemical work in progress by furnishing data on the nature of
hydrocarbon oxidation.
II. REVIEW OF THE LITERATURE
A. Respiratory Studies of Pseudomonas aeruginosa
Yaoi and Tamiya (1928, 1929) conducted a spectroscopic study
of various organisms and reported the presence of cytochrome in Pseudo
monas aeruginosa.They found components A,B,C and D in large concent
ration and in positions on the spectrum corresponding to those found in
yeast. Cytochrome C gave the most intense absorption band.
Friedheim (1951) found that the addition of pyocyanine to washed
cells of Pseudomonas aeruginosa increased their respiration in buffer
solution, but not in the presence of an oxidizable substrate. Respira
tion was inhibited by KCN and CO suggesting the importance of the cyto
chrome system for this organism.
Ehrismann (1954) reported that pyocyanine only slightly increased
the respiration of living bacteria and that the functioning of this
pigment was unaffected by HCN, CO, NaF and Na^AsO .
5
Yamagutchi (1954) found that M/2500 KCN was sufficient to inhibit
50-7 5 percent of the respiration of Pseudomonas aeruginosa.
Fujita and Kodama (1954) In a spectroscopic study of Pseudomonas
aeruginosa observed cytochromes A, B, C and D with B and C present in
greatest and equal intensities. An additional absorption band was
noticed within the range of 460 - 490 mjo.. Respiration was inhibited
49 percent by 0.0001M KCN but only 5 percent by CO. The effect of the
latter was reversed 2 percent by the addition of light. From this evid
ence, the authors conclude that CO sensitive cytochrome oxidase cannot
play an important role in the respiration of Pseudomonas aeruginosa.
Frei (1935) postulated that cytochrome and pigment both function
in the respiration of Pseudomonas aeruginosa.He suggested two altern
ate possibilities:
1. Hydrogen of substrate - cytochrome - pigment - oxygen
2. Hydrogen of substrate - pigment - cytochrome - cytochrome
oxidase — oxygen
Yamagutchi (1957) in studying the oxidation of various phenolic
compounds by Pseudomonas aeruginosa postulated the functioning of
cytochrome A, B, C and cytochrome oxidase and a CO insensitive
carrier in their oxidation.
B. Pissimilation of Glucose and Hydrocarbons by Pseudomonas aeruginosa
Aubel (1921) claimed that Pseudomonas aeruginosa formed ethyl
alcohol, acetic and formic acids as a result of glucose oxidation.
Pervoavanskii (1939) found that numerous strains of fluorescent
bacteria oxidized sugars directly without splitting the carbon chain.
Be isolated gluconic and 2 - keto gluconic acids as chief products
of the oxidation.
Lockwood, Tabenkin and Ward (1941) studied ten different species
Pseudomonas and found gluconic acid as an intermediate in glucose
oxidation. The final product was 2 - keto gluconic acid which account
ed for 67 percent of the total glucose consumed.
Barron and Friedemann (1941) found that the R. Q. of Pseudomonas
aeruginosa on glucose was 1.02, indicating complete oxidation. The
presence of 0.02 M NaF had no noticeable effect but 0.001 M iodo-
acetic acid and 0.005 M HCN greatly inhibited glucose breakdown.
Stone, Fenske and White (1940, 1942) reported that some members
of the genus Pseudomonas and other gram negative rods readily attack
ed light oils with an R. Q. of approximately 0.65. Heavier oils -were
less readily utilized and gave lower C02 / 0g ratios.
Bushnell and Haas (1911) found the R. Q. of several species of
Pseudomonas on petroleum fractions to vary from 0.50 - 0.70. There
seemed to be no direct correlation between R. Q. and. the nature of
the hydrocarbon attacked.
Strawinski and Stone (1942, 1945) isolated ortho — salicylic
acid from the fermentation of naphthalene by Pseudomonas aeruginosa..
Johnson, Goodale and Turkevicb (194?) conducted respiration
studies on 1 Bacillus aliphaticum*, en organism first described as
attacking hydrocarbons by Tausz and Peter (1919). It has since been
considered a. member of the genus Pseudomonas (Bushnell and Haas, 1941)
Honane and dodecane were completely oxidised by this organism as indie
ated by a C0g / Og ratio of 0.65 for both substrates. Incomplete oxida
tion was obtained with heptane and octane which gave ratios of 0.47
and 0.48 respectively. Glucose, used as a. basis of comparison, yielded
an R. Q. of 0.88. Respiration on glucose, octane and nonane was inhib
ited by 0.0008 M KCN and 0.25 M urethan. The authors claim that the
the final transfer of hydrogen in the oxidation of both glucose and
hydrocarbons is accomplished by cyanide sensitive cytochrome oxidase.
III. GLUCOSE AND HYDROCARBON OXIDATION IN THE WARBURG RESPIROMETER
A. Experimental Procedure
!• Technique and Substrates Employed
To investigate the oxidative mechanism of Pseudomonas aeruginosa,
Og uptake and COp evolution of the organisms were measured on glucose
and hydrocarbons. These determinations were accomplished using the
Warburg manometric technique according to Dixon (1954). Cetane,
CH5(CHg)Q_^_CHg, and naphthalene, G-^qHq, were' chosen for comparison
with glucose, because they represent an aliphatic end an aromatic
hydrocarbon and are very readily utilized'by strains of Pseudomonas
aeruginosa. (Strawinslci and Stone, 1940, 1942).
The Organisms and the Method of their Cultivation
Two strains of Pseudomonas aeruginosa were employed in this
studyj IIIC, isolated from cetane, IN, from naphthalene oxidation.
These strains were cultivated in one liter quantities of either gluc
ose broth or a hydrocarbon mineral salts medium. The gluedse broth
consisted per liter of 5 g. beef extract, 10 g. dextrose and 5 g. NaCl,
adjusted to pH 6.5. The hydrocarbon medium was designed in this labor
atory for the isolation of organisms capable of attacking petroleum.
(Strawinski and stone, 1940, 1942) . As employed in this investiga
tion, each liter contained 100 ml. of a mineral salt solution, tap
water to volume and one percent of either cetane or naphthalene added
after sterilisation. The salt mixture, prepared in large quantities
as a stock solution, consisted of 25 g. EH^NOg, 20 g. NagHPO^, 20 g.
KEgPO^, 5 g. MgSO^, 2 g. MnCl£, 0.5 g. FeClg per liter and traces of
KI, CuSO^ and ZnSO^.
The culture medium chosen at any given time depended on the sub
strate under investigation in the Warburg apparatus. Cetane and gluc
ose respiration was studied employing IIIC. This culture readily
attacks either glucose or cetane in the Warburg respirometer when
grown on cetane mineral salts. However, if glucose broth, is used as
the culture medium, glucose can be utilized but not cetane. Where
comparisons were to be made on glucose, the organisms were grown in
glucose broths where cetane alone or both cetane and glucose were to
be investigated, a, cetane mineral salts medium was necessarily
employed. Culture. IN attacks naphthalene in the Warburg apparatus
whether grown on glucose or naphthalene mineral salts broth. It was
difficult to obtain sufficient cells with the latter medium as the
organisms adhered to the insoluble naphthalene crystals. Glucose
broth was therefore employed as the culture medium throughout the
naphthalene investigation.
Glucose broth cultures were incubated for three days at 50°C.,
those on hydrocarbon media for one week. The glucose broth was centri
fuged directly, the hydrocarbon mineral salts medium was first decant
ed to remove all insoluble material. A Sharpies Super - Centrifuge,
operated at 50,000 revolutions per minute, was used to free the
organisms from their culture medium. One gram of the centrifuged
