Title: Effects of specific factors on the fatty acid composition of Streptococcus lactis
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Title: Effects of specific factors on the fatty acid composition of Streptococcus lactis
Physical Description: vi, 146 leaves : ill. ; 28 cm.
Language: English
Creator: Kral, Timothy Alan, 1951-
Copyright Date: 1978
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Subject: Lactococcus lactis   ( lcsh )
Fatty acids -- Analysis   ( lcsh )
Microbiology thesis Ph. D
Dissertations, Academic -- Microbiology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
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Statement of Responsibility: by Timothy Alan Kral.
Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 142-145.
General Note: Typescript.
General Note: Vita.
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Volume ID: VID00001
Source Institution: University of Florida
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oclc - 04244674
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EFFECTS OF SPECIFIC FACTORS ON THE
FATTY ACID COMPOSITION OF Streptococcus
lactis










By

Timothy Alan Kral


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY



UNIVERSITY OF FLORIDA

1978















ACKNOWLEDGMENTS


The author is extremely grateful to Dr. Kenneth L. Smith for his

guidance and support during the period of this research.

Gratitude is also expressed to Drs. Kermit C. Bachman, Arnold S.

Bleiweis, and Paul H. Smith for serving as members of the supervisory

committee, and to all the members of the Department of Microbiology

and Cell Science and the Dairy Science Department for their assistance.

Special thanks go to the author's parents, Mr. and Mrs. George E.

Kral for their support, encouragement, and love. This work is dedicated

to them.


















TABLE OF CONTENTS




ACKNOWLEDGMENTS . . . . . . . . .. . . . . ii


ABSTRACT . . . . . . . . . . . . .


S v


INTRODUCTION . . . . . . . . ... . . . . . 1

ORGANISM . . . . . . . . . . . . . . 5


COMMON METHODS . . . .
Growth Conditions . . ...
Lipid Extraction and Esterification
Fatty Acid Methyl Ester Analysis


CHAPTER I. EFFECTS OF GROWTH TEMPERATURE ON THE FATTY ACID
COMPOSITION OF Streptococcus Zacti . . .
Literature Review . . . . . . . . . .
Additional Methods . . . . . . . . . .
Results . . . . . . . . . .
Discussion . . . . . . . . . . . .


CHAPTER II. EFFECTS OF pH
Streptococcus
Literature Review . .
Additional Methods .
Results . . . .
Discussion . . .


ON THE FATTY ACID
lactis . . .


CHAPTER III. EFFECTS OF GROWTH PHASE ON THE FATTY ACID
COMPOSITION OF Streptococcus lactis . . .
Literature Review . . . . . . . . . . .
Additional Methods . . . . . . . . . .
Results . . . . . . . . . . . . .
Discussion . . . . . . . . . . . .

CHAPTER IV. EFFECTS OF VARIOUS CARBOHYDRATE SOURCES ON THE
FATTY ACID COMPOSITION OF Streptococcus lacti .
Literature Review . . . . . . . . . . .
Additional Methods . . . . . . . . . .
Results . . . . . . . . . . . . .
Discussion . . . . . . . . . . . .








iii


. . 10
. . 10
. . 12
. . 13
. . 31


COMPOSITION








CHAPTER V. EFFECTS OF CITRATE ON THE FATTY
ACID COMPOSITION OF Streptococcus lactis . . . . 98
Literature Review . . . . . . . . ... . . .98
Additional Methods . . . . . . . . . . . . 98
Results . . . . . . . . ... . . . . . . 99
Discussion . . . . . . . . ... . . . .. .117

CHAPTER VI. EFFECTS OF ADDED FERMENTATION PRODUCTS ON THE
FATTY ACID COMPOSITION OF Streptococcus lactis . .. .118
Literature Review . . . . . . . . .. . . .118
Additional Methods . . . . . . . . ... . . 119
Results . . . . . . . . ... . . . . . .119
Discussion . . . . . . . . ... . . . . .137

GENERAL DISCUSSION . . . . . . . . ... ..... .138

BIBLIOGRAPHY . . . . . . . . ... . . . . 142

BIOGRAPHICAL SKETCH . . . . . . . . ... ..... .146















Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy



EFFECTS OF SPECIFIC FACTORS ON THE
FATTY ACID COMPOSITION OF Streptococcus
lactis


By

Timothy Alan Kral

August 1978

Chairman: Kenneth L. Smith
Major Department: Microbiology and Cell Science

The effects of temperature, pH, growth phase, carbohydrate source,

citrate, and fermentation products on the fatty acid composition of

Streptococcus lactis ATCC 12929 were investigated. Organisms were grown

in a defined medium at Constant temperature and pH. Optical densities

of cultures were measured throughout growth to determine doubling times.

At the appropriate time during growth, cells were harvested, lipids were

extracted, and fatty acids were converted to methyl esters. The methyl

esters were analyzed via gas chromatography.

The major fatty acids found were 14:0, 16:0, 16:1, 18:0, 18:1, &19:0,

and 21:0. The most dramatic differences occurred in the 18:1 and A19:0

fatty acids under all conditions tested. At a temperature of 300C,

18:1 levels were their highest while A19:0 levels were their lowest.

As the temperature became more adverse in either direction, the A19:0

fatty acid increased at the expense of decreases in the 18:1 fatty acid.








A pll of 6.5 resulted in a high level of 18:1 fatty acid with a very low

level of the A19:0 fatty acid. At low pH values, the A19:0 levels

again increased with decreasing levels of 18:1 fatty acid. At higher

pH values, the 18:1 content decreased while the A19:0 content remained

low. Levels of the 18:1 fatty acid also decreased with concomitant

increases in the A19:0 fatty acid as cells proceeded from exponential

to stationary phase of growth.

Growth on various carbohydrate sources demonstrated variations in

18:1 and A19:0 fatty acid levels with no quantitative correlation with

growth rate. Varying concentrations of citrate caused insignificant

changes in the levels of these two fatty acids. Only at a level of

citrate ten times that normally found in the growth medium were levels

of A19:0 elevated and 18:1 levels depressed. In all cases of added

fermentation products, higher concentrations of those products caused

lower levels of the 18:1 fatty acid and higher levels of the A19:0

fatty acid.

In general, when the organisms seemed to be most content, the

levels of 18:1 fatty acid would be high while levels of A19:0 fatty

acid would be low. As conditions became more adverse, the A19:0

fatty acid increased at the expense of decreasing 18:1 fatty acid levels,

except at pH values above 6.5 where A19:0 levels remained low. Al-

though a gross relationship between growth rate and A19:0 fatty acid pro-

duction seemed apparent, mathematical models tested could demonstrate

no quantitative correlation.





Chairman















INTRODUCTION


Every living organism differs from its environment in both its phy-

sical properties and its chemical composition. In order to maintain

these differences, some structure must exist which separates the organism

from its surroundings. This structure is the cell membrane. But

separating the cell from its environment is only one role of the cell

membrane. Since the cell must interact with its environment, the

cell membrane must allow selective interaction between the interior

of the cell and its surroundings (11).

Since biological membranes play a crucial role in almost all cellular

phenomena, an understanding of the molecular organization of the mem-

brane is necessary. The principal components of membranes are lipids

and proteins. Carbohydrates may be present in small amounts, usually

from none to less than 10% of the total mass of the membrane (1). The

ratio of lipid to protein varies greatly from one type of cell to an-

other. In the myelin membrane sheathing of nerve fibers the proportion

of lipid to protein is approximately nine to one. This is probably due

to the fact that the myelin membrane functions as an insulator. Mito-

chondrial membranes, on the other hand, have a ratio of about one to

one. This relatively high protein ratio is accounted for by the

many enzyme systems associated with the mitochondrion (4).

Proteins play a variety of roles in membranes. They may contribute

to the structural integrity of the membrane, they can act as enzymes

or they can function as pumps, transporting molecules in and out of








tie cell (2). It is believed that it Is the interaction of these

proteins with the lipid components which gives the membrane its

characteristics and vital properties.

A necessary step toward the understanding of membranes is the

understanding of the lipid component. Organisms which have many advan-

tages for studying membrane lipids are the bacteria. They have no

intracellular membranes, and their lipid compositions are much simpler

than the compositions of eucaryotic cells (8). Of the bacteria, the

gram positives are less complex than the gram negatives since gram

positives have one cytoplasmic membrane while gram negatives have an

inner and outer membrane (33).

Phospholipids are the principal lipid constituents of bacterial

membranes. The common lipids found are phosphatidyl serine, phospha-

tidyl ethanolamine, phosphatidyl glycerol and cardiolipin (41). While

most gram negatives are composed almost entirely of phospholipids,

most gram positives also include varying proportions of glyco- and

neutral lipids. Sphingolipids and cholesterol are absent from bacteria

(41). Membrane lipids differ among themselves not only in polar head

groups, but also in the identity of their constituent fatty acids.

Phospholipid and glycolipid molecules may contain any two of more than

half a dozen fatty acids while a neutral lipid may contain any three

(4).

Phospholipids and glycolipids, the two most common lipids in gram

positive bacteria, have a head and two tails, the head being the phos-

phate moiety or carbohydrate respectively, and the tails being the

fatty acid chains. The tails, being hydrophobic, tend to point away

from water, while the head groups, being hydrophilic, are attracted

to water. Lipids in membranes are arranged to accommodate this








amphipathic character. They form a bllnyer, two layers back to back,

with their hydrophilic heads constituting the outer surfaces and their

hydrophobic tails buried in the interior (2). This bilayer structure

results in maximizing both hydrophobic and hydrophilic interactions

which contribute to the stabilization of the membrane (44).

These thermodynamic considerations along with experimental results

accommodate a mosaic membrane structure in which globular proteins

alternate with sections of the bilayer in the cross-section of the

membrane. These globular proteins, being amphipathic to varying degrees,

would fit well into the bilayer, with hydrophobic amino acid portions

associating with the hydrophobic fatty acid interior and the hydro-

philic portions associating with the hydrated surface (44).

This mosaic membrane structure is by no means static. Both the

lipids and the proteins have considerable freedom of movement (2).

This is due to the fact that under physiological conditions, the

lipids are in a fluid rather than a crystalline state (44). This

fluid nature of membranes has been established by application of -both

x-ray diffraction and differential scanning calorimetry to bacterial

membranes. These techniques have shown that the membrane can pass

through a thermal phase transition. At temperatures below the transi-

tion, the fatty acids are in a relatively rigid crystalline state while

at temperatures above the transition, the fatty acids assume a more

fluid random structure. At temperatures in between, both fluid and

solid lipid phases exist. The temperature range of this transition is

determined largely by the fatty acid composition of the membrane.

Chain length, degree and position of unsaturation, and the configuration

of the unsaturation (cis or trans) of the fatty acids are important

factors. Longer chain lengths lead to greater stability of the crystalline








state and thus to higher transit on temperatures (4,41). Membranes

formed from ipirds contalnlAng fully saturated linear fatty acids also

have higher transition temperatures. Lipids with cis-unsaturated fatty

acids undergo transitions at much lower temperatures due to the instability

created by the discontinuity in the regular array of fatty acid side

chains (18,41).

Organisms have a sufficient number of double bonds in their lipids

to maintain the bilayer membrane in a lipid crystalline or fluid state

at growth temperatures (18,41). Lipids of some organisms contain,

instead of double bonds, cyclopropyl groups or branched chains which

produce kinks in the regular array of fatty acid side chains, and serve

to maintain membrane fluidity (18). This is especially true in gram

positive bacteria (23,34,35). This seems to be necessary since such

processes as membrane biogenesis, transmembrane transport, and exo- and

endocytosis can occur only when the membrane exhibits sufficient fluidity.

If a membrane is too fluid, it may lack integrity. Therefore, living

organisms have evolved mechanisms to regulate the degree of unsaturation

as well as chain length, ensuring a proper degree of membrane fluidity

(6,41).

The regulation of proper fatty acid composition when the organism

is subjected to a variety.of environmental conditions is the theme

of this work. The organism chosen for this investigation was Strepto-

coccus lactis ATCC 12929, a gram positive bacterium. Six environmental

parameters were chosen: temperature, pH, growth phase, carbohydrate

source, presence of citrate, and presence of added fermentation products.















ORGANISM


S. lactis ATCC 12929 is a gram positive coccus which typically

occurs in pairs. Antigenically, it is a Lancefield Group N bacterium.

Its growth range is from below 100C to 400C with an optimum at about

30C. Its pH range is from about 4.2 to 9.2. In broth culture, it

grows microaerophilically, while on milk agar it forms pinpoint size

colonies. S. lactis is probably of plant origin, but it is found

commonly as a contaminant in milk and is used industrially in the

production of dairy products such as buttermilk (12).

S. lactis ATCC 12929 contains a variety of fatty acids, some of

which occur in relatively large quantities, while others are only

found in trace amounts. These are listed in Table 1. This dissertation

will be concerned only with the major fatty acids.














Table 1. Fatty acids found in S. Zactis ATCC 12929.



Major fatty acids (>1.0%) Trace fatty acids (<1.0%)




14:0 12:0

16:0 12:1

16:1 14:1

18:0 iso 16:0

18:1 anteiso 21:0

A19:0 22:0

21:0















COMMON METHODS


The methods mentioned here were used in all of the experiments

with some alterations in individual experiments. Those alterations

will be noted when necessary.


Growth Conditions

S. lactis ATCC 12929 was propagated in a defined medium (40)

supplemented with 1.0% glucose. A 2.0 ml inoculum of an overnight culture

(300C) was added to 200 ml of the same medium in a 1.0 1 jar.

Immediately prior to inoculation, the medium was sterilized by mem-

brane filtration. The jar was kept immersed in a constant temperature

water bath held at 30C. Constant agitation was maintained by a

magnetic stirrer in the jar. Immersed in the medium were two pH

electrodes which were connected to a Versatrol Control Unit (Assembly

Products, Inc.) through a pH meter (Beckman). The Versatrol controlled

a Model 610 Randolf Pump equipped with a variable speed control unit.

As the pH decreased due to acid production by the bacteria, the

Versatrol Control Unit automatically activated the pump which fed

sterile 3N KOH into the growth vessel. In this manner, the pH was

maintained at 6.5. An aquarium pump forced air through two sterile

cotton filters into the growth vessel, maintaining a constant positive

pressure, thus allowing for a contaminant-free system. Figure 1

shows a diagram of this growth setup. Culture purity was verified at

the end of each experiment by the gram stain technique. Samples were


7


















Control Unit


Magnetic Stirrer



Figure 1. Diagram of the growth setup.








taken from the cultures at various times during growth, and optical

densities were measured at 540 nm using a Spectronic 20 (Bausch and

Lomb) in order to construct growth curves and thereby determine

doubling times. When the cultures reached mid to late exponential

phase, they were immediately inactivated by addition of 5.0% trichloro-

acetic acid (w/v).


Lipid Extraction and Esterification

The trichloroacetic acid-treated samples were pelleted in a Sorvall

Superspeed RC2-B centrifuge at 12,000Xgfor10 minutes. The pellet

was extracted with chloroform:methanol (2:1v/v) according to the method

of Kanfer and Kennedy (24). The extract was evaporated under nitrogen

gas and then transesterified with 2.0% H2S04 in methanol (w/v) for 2 hours

at 80C. Fatty acid methyl esters were extracted twice into pentane

and analyzed via gas chromatography.


Fatty Acid Methyl Ester Analysis

The pentane extracts were concentrated using nitrogen gas and 0.5ul

aliquots were injected into a Series 810 F & I Research Chromatograph

equipped with a flame ionization detector and a 183 cm by 0.64 cm

stainless steel column. The column was packed with 10% EGSS-X on Gas

Chrom Q (100/120 mesh). The gas chromatograph was run isothermally at

180C. Nitrogen gas was used as the carrier at a flow rate of 30 ml/min.

Fatty acid methyl esters were identified by comparison with authentic

standards (Applied Sciences Laboratory) as well as by relativeweight percent-

ages. Relative percentages of fatty acid methyl esters were determined

by the product of retention time and peak height (3).















CHAPTER I
EFFECTS OF TEMPERATURE ON THE FATTY
ACID COMPOSITION OF S. Zactis



Literature Review

As mentioned in the introduction, the mosaic membrane structure is

not static. The lipids and proteins have considerable freedom of move-

ment due to the fact that under physiological conditions, the lipids

are in a fluid rather than a crystalline state (2,44). With regard to

temperature, the general rule is that as the growth temperature is

lowered, the composition of the fatty acid chains is modified in the

direction of the lower average melting points. This adjustment makes

it less likely that all of the lipids will become frozen, a condition

that would interfere with vital functions (27). Changes in the physical

state of the lipids affect biological activities in membranes at two

characteristic temperatures. Below the lower temperature, all the mem-

brane lipids are in a solid state. Above the higher temperature, all

of the membrane lipids are in a fluid state (49). At temperatures

between these low and higher temperatures, both fluid and solid phases

exist. The temperature range of this transition is determined largely

by the fatty acid composition of the membrane (41). Membranes containing

fully saturated linear fatty acids have higher transition temperatures

whereas membranes containing cis-unsaturated fatty acids undergo transi-

tions at much lower temperatures due to the instability created by the

discontinuity in the regular array of fatty acid side chains (18,41).








In gram negative bacteria, as the temperature is lowered, there is

usually an increased proportion of unsaturated fatty acids at the expense

of saturated fatty acids (27). Marr andIngraham (36) demonstrated

that the proportion of unsaturated fatty acids in Eacherichia coli in-

creased continuously as the growth temperature was decreased in both

minimal and complex media. The proportion of cyclopropane fatty acids

also decreased at lower temperatures. Similar changes were observed

when Scrratia marcescens was grown at 30C and 100C (26).

In Pseudomonas fluorescens, degree of fatty acid saturation varied

to some extent but not over the whole growth temperature range. A

minimum degree of saturation was obtained at about 100C and further

decreases of the growth temperature did not significantly alter the fatty

acid composition. P. fluorescens controls its fatty acid com-

position only over the lower third of its growth temperature range by

increasing its relative amount of unsaturated fatty acids with de-

creasing temperature. A precise degree of saturation apparently is not

required for normal growth at higher temperatures (10,19).

In some gram positive bacteria there are increased amounts of

lower melting branched chain fatty acids at the expense of the higher

melting branched and straight chain fatty acids at lower growth temper-

atures. In addition, a fatty acid desaturating system is induced in

some bacilli at lower growth temperatures resulting in lower melting

unsaturated fatty acids (27). In both Bacillus caldolyticus and Bacillus

caldotcnax, decreased growth temperature caused an increase in iso 15:0

and a decrease in iso 17:0. The iso 17:0 would correspond to a more

saturated fatty acid due to its higher melting point. Also observed

was an increase in the iso 16:0 fatty acid corresponding to a decrease








in the 16:0 fatty acid (48). Even in E. coli, a gram negative bacterium,

the cis-unsaturated fatty acids can be totally replaced by branched chain

fatty acids bearing bulky substituents which disrupt membrane packing

(42).

Many gram positive bacteria, however, have fatty acid profiles

similar to gram negative bacteria and behave accordingly with respect

to changes in growth temperature. Clostridium butyricum shows a general

increase in the proportions of unsaturated and cyclopropane fatty acids

when the temperature is lowered from 37C to 25C (27). Decreases in

growth temperature have also resulted in increased ratios of unsaturated

to saturated fatty acids in Streptococcus mutans (17).

The fatty acid composition of S. lactis 527, grown at two different

temperatures (100C and 30*C), has also been investigated. Results

showed that unsaturated fatty acids increased while lactobacillic

acid (A19:0) decreased at 100C compared to 300C in the neutral lipid

fraction. Complex lipids showed little changes in fatty acids with

respect to temperature differences. The investigators postulated that

the formation of the lactobacillic acid might have been delayed or

inhibited at the lower temperatures (47).

Lactobacillic acid has also been shown to decrease with lower growth

temperature in Streptococcus faecalis. The final percentage of lacto-

bacillic acid in cells grown at 27, 37, and 470C was 15.9, 22.6, and

36.4% respectively (22).


Additional Methods

Organisms were grown at 10, 15, 20, 25, 30, 35, and 390C.

Growth curves were plotted and doubling times were determined for

growth at each temperature. This experiment was run in triplicate and

an analysis of variance was applied to the data.










Results

Doubling times for growth at the various temperatures are listed in

Table 2. The fastest growth occurred at 35C under the given set of

conditions.

Table 3 lists the percentages of the fatty acids at each temperature

tested. Except for the 18:1 and A19:0 fatty acids, the remaining fatty

acids showed relatively small differences throughout the temperature

range. The 14:0 content remained constant at 100, 15", and 20C, and

then showed a gradual rise of about 6 percentage points from 20 to 390C.

A gradual rise of 5 percentage points occurred in the 16:0 content from

100 to 35%C at which point it remained constant up to 390C. The 16:1,

18:0, and 21:0 fatty acids showed slight increases and decreases of no

more than 3 percentage points throughout the range tested.

Dramatic differences appeared in the 18:1 and A19:0 fatty acid

contents. An increase of approximately 20 percentage points occurred

in the 18:1 fatty acid going from 10 to 30C followed by a sharp

decrease of 30 percentage points from 30 to 390C. The opposite effect

was observed in the A19:0 fatty acid. Going from 100 to 30C, a drop

of 23 percentage points occurred followed by a rise of 20 percentage

points when the temperature increased from 300 to 39C. These differences

are graphically represented in Figures 2-8.

F-values and significance of differences are shown in Table 4.

Significant differences were seen in all of the fatty acids except in

the 18:0 fatty acid.
















Table 2. Doubling times of S. tactis grown at various temperatures.



Temperature (C) Doubling time (min)




10 3800

15 2100

20 465

25 190

30 75

35 70

39 312
















Table 3. Fatty acid percentages from S. lactis grown at various
temperatures.



Temperature (OC)

Fatty acid 10 15 20 25 30 35 39




14:0 1.9 1.7 1.9 2.7 3.5 5.0 7.1

16:0 27.8 29.3 30.3 31.6 33.0 33.7 33.1

16:1 5.5 4.6 3.8 3.6 2.7 3.1 4.4

18:0 1.7 2.5 1.7 2.0 2.9 2.0 2.4

18:1 28.2 35.3 41.7 42.6 46.7 38.1 18.7

A19:0 30.3 23.1 16.9 14.0 8.1 12.7 28.2

21:0 2.9 1.9 1.5 2.0 1.6 2.7 3.6



































Figure 2. Percentages of the 14:0 fatty acid from S. lactis grown at
various temperatures.





17






60







50






40







30






20






10







0
10 15 20 25 30 35 40

Temperature (C)




































Figure 3. Percentages of the 16:0 fatty acid from S. lactis grown at
various temperatures.































































10 15 20 25

Temperature (OC)


30 35 40


50 t


40 1


10 1


" F~ -



































Figure 4. Percentages of the 16:1 fatty acid from S. Zactis grown at
various temperatures.















































10 15 20 25
Temperature (*C)


60 .


50 .


40 r


10 L


30 35 40


Qy~





































Figure 5. Percentages of the 18:0 fatty acid from S. lactis grown at
various temperatures.





























































10 15 20 25

Temperature (C)


30 35 40


60 t


50 P


40


Lp
S 30
a,
pt


20 r


r ~Lr r


--~Y-- ~--- --



































Figure 6. Percentages of the 18:1 fatty acid from S. lactis grown at
various temperatures.
































































10 15 20 25

Temperature ("C)


30 35 40


30 P


10 I


"~------ '-



































Figure 7. Percentages of the A19:0 fatty acid from S. lactis grown
at various temperatures.





































































10 15 20 25

Temperature (C)


30 35 40


50 I


40 r


S30
u
ar
U,


20 r


10 r


- ----




































Figure 8. Percentages of the 21;0 fatty acid from S. Zactis grown at
various temperatures.





29






60






50







40







30






20







10






0
10 15 20 25 30 35 40

Temperature (C)
















Table 4. F-values and significance of differences for temperature
experiment.



Fatty Acid F-valuea Significanceb




14:0 58.43 *

16:0 17.37 *

16:1 10.72 *

18:0 2.12 NS

18:1 52.50 *

619:0 107.02 *

21:0 11.06 *



a F 2.85
a .05,6,14 285

= significant; NS = not significant










Discussion

Four of the seven major fatty acids, 14:0, 16:0, 16:1, and 21:0

showed statistically significant differences, but the maximum range of

differences (<6 percentage points) seemed so small that on first analysis,

any functional significance seemed unlikely. Only the 18:1 and A19:0

fatty acids showed statistically significant differences as well as

relatively large numerical differences. The 18:0 fatty acid showed no

significant differences at all.

It probably can be assumed that S. lactis, as well as all forms of

life, must maintain a functional membrane fluidity to maintain the

essence of life. If that functional fluidity is indeed derived from

the balances of saturated and unsaturated fatty acids in the bilayer of

the membrane, then it is difficult to envisage how the dramatic differ-

ences in the 18:1 and A19:0 fatty acids could account for that level of

fluidity. Rather than increase or decrease continuously throughout

the temperature range, each achieved a point (30C) where it changed

directions. The 18:1 fatty acid increased from 10 to 30C and then

decreased sharply up to 390C. The opposite effect is seen in the

A19:0 fatty acid. There seems to be a balance between the two, which is

reasonable since the A19:0 fatty acid is derived from the 18:1 fatty

acid by the methylation of the 18:1 double bond by S-adenosylmethionine.

This reaction is catalyzed by cyclopropane fatty acid synthetase (37,50).

Summing up the 18:1 and A19:0 percentages at each temperature generates

the values seen in Table 5. These sums also represent the amounts of

18:1 fatty acid incorporated into the membrane. As the temperature pro-

ceeds from 10 to 390C, the sums remain constant up to 20C and then

consistently decrease by about 11 percentage points. If the A19:0















Table 5. Sums of the 18:1 and
growth temperature.


A19:0 fatty acid percentages at each


Temperature (OC) 18:1 A19:0 18:1 + A19:0




10 28.2 30.3 58.5

15 35.3 23.1 58.4

20 41.7 16.9 58.6

25 42.6 14:0 56.6

30 46.7 8.1 54.8

35 38.1 12.7 50.8

39 18.7 28.2 46.9









fatty acid is considered comparable to the 18:1 fatty acid in regards

to fluidity characteristics due to its cyclopropane ring which would

cause some disorder in the fatty acid packing, then the decrease in

sums would represent a decrease in total unsaturation which is consistent

with the theory mentioned previously. Accordingly, the small increases

in the 14:0 and 16:0 fatty acid percentages, approximately 11 percentage

points, would both balance the decrease in unsaturation as well as

satisfy the theory: with increasing temperature, there is an increase

in saturation with an accompanying decrease in unsaturation.

But why do the percentages of the 18:1 and A19:0 fatty acids change

so drastically? Changes to conserve fluidity obviously could have been

more conservative. There must be another reason to explain the differ-

ences observed. Both the 18:1 high point and the A19:0 low point occur

at 30C. In this experiment, the most rapid growth occurred at 35C, but

the optimum temperature usually quoted for S. lactis is around 30"C (12).

Whether the optimum growth temperature corresponds to the fastest growth

temperature is a matter of semantics. In any case, S. Zactis seems to

grow quite well at 30C. Assuming that S. lactis is "happiest" at 30"C,

then temperatures above and below 300C would be less desirable. This

corresponds to the differences observed in the 18:1 and A19:0 fatty acids.

As the temperature proceeds away from 30C, the 18:1 content decreases while

the Al9:0 content increases. Since the conversion of 18: to A19:0 occurs

to the greatest extent when the organisms encounter adverse temperatures,

it seems likely that the A19:0 fatty acid may be protective in some way.

















CHAPTER II
EFFECTS OF pH ON THE FATTY ACID COMPOSITION
OF S. lactis



Literature Review

Very little work has been done correlating fatty acid differences

with differences in pH. In 1975, Drucker, Griffith and Melville (16)

reported on the effects of pH on the fatty acid profiles of various

strains of S. mutans. Cells were grown at a series of pH values ranging

from 5.8 to 8.3. Below pH 6.5 fatty acid profiles were relatively

stable. From pH 6.7 to 7.3 the proportions of 16:1 and 18:0 changed.

The 16:1 increased approximately 10 percentage points while the 18:0

decreased about 12. These same fatty acids remained stable from pH

7.3 to 8.3. The 16:0 and 18:1 fatty acids remained relatively constant

below pH 7.0, while increasing and decreasing randomly at alkaline pH

values. Therefore, the major cellular fatty acids of the strains of

S. mutans investigated remained fairly constant below pH 6.7, while

showing significant differences above pH 6.7 without, however, demon-

strating any discernible patterns.

Effects of pH on the fatty acid composition of Bacillus acidocaldarius

have also been examined. Results indicated that the effects of temper-

ature and pH are interdependent. At pH 2.0, increasing temperature

raised the proportion of iso and anteiso fatty acids (as much as 9

percentage points) while at pH 5.0, the effect of increasing temperature

was reversed and the proportion of cyclohexyl fatty acids was increased

(as imich as 22 ]perclCenLIg, point) (14).

34(









Studies done with S. faecalis showed that low pH was correlated with

an increased synthesis of cyclopropane fatty acids (21).

In E. coli, below pH 6.2, the percentage of the A17:0 fatty acid

increased with acidity until at pHl 4.5, it was three times that at pH 6.2.

There was little change in A17:0 above pH 6.2. Below pH 5.5, some 619:0

fatty acid also appeared. And there was a decrease in the 16:1 fatty

acid with increasing acidity below pH 6.2 (29).


Additional Methods

The organisms were grown at pH values of 4.5, 5.0, 5.5, 6.0, 6.5,

7.0, 7.5, 8.0, 8.5 and 9.0. Growth curves were plotted and doubling

times determined for growth at each pH. This experiment was run in

triplicate and an analysis of variance was applied to the data.


Results

Table 6 lists the doubling time:; for growth at each pH tested. A pH

of 6.0 seemed to induce the most rapid growth.

Fatty acid percentages for each pil tested are listed in Table 7. All

of the fatty acids in this experiment demonstrated differences with the

18:1 and 619:0 fatty acids again showing the really dramatic differences.

The most constant fatty acid was 16:1 showing a gradual increase of

5 percentage points going from pH 4.5 to 9.0. The 14:0 fatty acid

showed an increase of 5 percentage points from pH 5.0 to 6.0, then a

slight decrease of 2 percentage points at pH 7.0, and then a gradual

increase of 5 percentage points up to pH 9.0. A fairly constant per-

centage with a 2 percentage point dip at pH 6.5 and an increase of 4

percentage points going from pll 8.5 to. 9.0 characterized the 18:0 fatty

acid. The 21:0 content also remained constant from pH 5.5 to 9.0, but

from 5.5 to 5.0 it Increased 7 percentage points and then leveled out















Table 6. Doubling times of S. Zactis grown at various pH's.



pH Doubling time (min,)

























O -h 3 r iH 0






co rO 9' io -H 'D N
00 I
m .


o O O 0 N 0 I n





a o -o o rIn c-M N N












11 '0 N H o N
0























c' C') -4
C- '4 C') N C') 0)
- lo O










cC D '^O-4 CT')
C-t
co *



C!










u








*-i<- ^- ^l -l -l ^-l ^- c
ii3 TOl~









going down to pH 4.5. The 16:0 fatty acid decreased 9 percentage points

from pH 4.5 to 6.0, remained constant up to pH 7.5, and then increased

9 percentage points up to pH 9.0.

A very large increase of 48 percentage points was observed going

from pH 4.5 to 6.5 in the 18:1 fatty acid which then showed a decrease

of 20 percentage points as the pH increased to 9.0. The A19:0 fatty

acid, on the other hand, decreased 25 percentage points going from pH

4.5 to 5.5, and then continued to decrease very gradually about 10

percentage points from pH 5.5 to 9.0. Figures 9-15 demonstrate these

differences graphically.

The F-values, seen in Table 8, show significant differences for all

of the fatty acids.


Discussion

With changing pH, all seven of the major fatty acids demonstrated

statistically significant differences. Again the most dramatic numerical

differences were seen in the 18:1 and A19:0 fatty acids. Why the other

five fatty acids show any differences at all is purely up to conjecture.

If the proposal that the levels of 18:1 and A19:0 fatty acids correspond

to "happiness" in this bacterium, then the results observed do make

sense. Most rapid growth was observed at pll 6.0, very close to the

high point of 18:1 at pH 6.5. Again the "happiness" point may not

exactly correspond to most rapid growth. If the organism is "happiest"

at pH 6.5, then the decrease in 18:1 as the pH is increased or decreased

from that value would lend further support for the proposal that the

high point of 18:1 content corresponds to optimum growth conditions for

S. lactis.



































Figure 9. Percentages of the 14:0 fatty acid from S. Zactis grown at
various pH's.













60







50







40







30
cP





20







10







0
4.5 5.0 5.5 6.0 6.5 7. 7.5 8.0 8.5 9.0

pH



































Figure 10. Percentages of the 16:0 fatty acid from S. Zactis grown at
various pH's.




















50 r


40 r


30 F


4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0



































Figure 11. Percentages of the 16:1 fatty acid from S. lactis grown at
various pH's.





44






60






50







40







30

U





20







10







4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0




































Figure 12. Percentages of the 18:0 fatty acid from S. lactis grown at
various pH's.













60







50







40








30
u
u




20







10







0
4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0

pH



































Figure 13. Percentages of the 18:1 fatty acid from S. lactis grown at
various pH's.




















50 1


30 t


4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0

pHl



































Figure 14. Percentages of the A19:0 fatty acid from S. Zactis grown at
various pH's.










































30 1


10 p


Ut
0)


4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0



































Figure 15. Percentages of the 21:0 fatty acid from S. lactis grown at
various pH's.













60







50






40







u 30


ci




20






10







0
4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
















Table 8. F-values and significance of differences for pH experiment.


Fatty Acid


Significance


a F = 2.39
.05,9,20
b = significant
= significant


F-valuea


14:0

16:0

16:1

18:0

18:1

A19:0

21:0


9.60

6.98

29.87

4.79

152.32

75.55

23.39




54




As for the l19:0 fatty acid, as the pH decreased from pH 6.5, the

levels of L19:0 increased sharply, corresponding to an increase in

adverse conditions. But above pH 6.5, the A19:0 level remained fairly

constant and even decreased further at pH 9.0. Either the high pH is

effecting the activity of the cyclopropane fatty acid synthetase,

causing some sort of inhibition, or maybe the organism does not require

the hypothetical protection afforded by the A19:0 fatty acid at the

higher pH values.
















CHAPTER III
EFFECTS OF GROWTH PHASE ON THE FATTY ACID
COMPOSITION OF S. lactis



Literature Review

Fatty acid composition in bacteria has long been known to vary with

the phase of growth. In 1961 it was observed that Lactobacillus ara-

binosus harvested in stationary phase had higher proportions of lacto-

bacillic acid and lower levels of unsaturated fatty acids than cells

harvested during exponential phase (25). In culturesof E. coli, the

accumulation of cyclopropane fatty acids at the expense of unsaturated

fatty acids in stationary phase has also been reported (5,29). In

S. marcescens, low amounts of cyclopropane fatty acids and high

amounts of monoenoic fatty acids were present during early stages of

growth, the latter being converted almost completely to cyclopropane

fatty acids later in growth (26). The gram positive bacterium,

S. faecalis, showed similar effects with cyclopropanes reaching a

maximum level at the beginning of stationary phase (21,22). Analogous

changes in the proportions of the 18:1 and A19:0 fatty acids of Myco-

bacterium phei have also been observed during the growth cycle (26).

Apparently most organisms studied with respect to growth phase have

been those containing cyclopropane fatty acids, and in all cases cited

the results are the same cyclopropane fatty acids accumulate during

stationary phase at the expense of their unsaturated fatty acid precursors.










Additional Methods

Cells were grown in 1500 ml of medium in a 2.0 1 jar. Sterile air

was not pumped in since the 2.0 1 jar had a very good seal as evidenced

by culture purity at the end of the experiment. Samples of 150 ml were

taken from the growth vessel at various phases of growth by way of an

aspiration line from the growth vessel to a collection flask. Table 9

lists the times after inoculation that samples were collected as.well

as associated optical densities and the six phases of growth. This

experiment was run in quadruplicate and an analysis of variance was

applied to the data.


Results

The percentage distributions of the fatty acids at each phase of

growth are shown in Table 10. There was very little difference in

relative fatty acid percentages between early and mid exponential phases.

Both the iso 16:0 and 21:0 fatty acids remained at less than 1.0% of

the total during these phases. More dramatic differences occurred

when the cells reached late exponential and early stationary phases.

The iso 16:0 content increased to 2.3% in late exponential phase, while

the 21:0 content increased to 1.0% by early stationary phase. A de-

crease of nearly 12 percentage points was observed in the 18:1 content

going from early exponential to early stationary phase, while the A19:0

fatty acid showed an increase of more than 6 percentage points over the

same range. In late stationary phase, a further increase occurred in

the iso 16:0, A19:0 and 21:0 fatty acids, with a further decrease occurring

in the 18:1 fatty acids.

The 14:0, 16:0, 16:1 and 18:1 fatty acids showed very small differ-

ences throughout growth. The iso 16:0 and 21:0 fatty acids, although

















Table 9. Sampling times and corresponding optical densities and phases
of growth.



Time after Growth
Sample inoculation (hrs) O.D. (540 nm) phase





1 6.0 0.20 Early exponential

2 7.0 0.40 Mid exponential

3 8.5 0.80 Late exponential

4 11.0 1.30 Early stationary

5 16.0 1.30 Mid stationary

6 25.0 1.25 Late stationary


--















Table 10. Fatty acid percentages from S. lactis during various phases
of growth.



Exponential Stationary
Fatty Acid
Early Mid Late Early Mid Late

14:0 3.4 3.3 3.8 5.1 5.0 4.9

iso 16:0 <1.0 <1.0 2.3 2.4 3.3 3.5

16:0 30.2 29.6 29.3 31.1 29.7 28.9

16:1 2.1 2.0 2.1 2.6 2.5 2.6

18:0 3.5 2.5 3.0 2.9 3.5 3.8

18:1 57.4 58.0 53.0 45.6 42.0 39.7

A19:0 2.1 3.0 5.1 8.4 11.7 12.1

21:0 <1.0 <1.0 <1.0 1.0 1.5 1.6








increasing from trace amounts in the initial phases to a few percent in

the later phase, still only showed relatively minor differences. Only

the 18:1 and A19:0 fatty acids showed relatively large differences.

These differences are graphically represented in Figures 16-23.

The F-values, listed in Table 11, indicate that the 14:0, iso 16:0,

18:1, A19:0 and 21:0 fatty acids demonstrate significant differences while

the 16:0, 16:1 and 18:0 fatty acids do not.


Discussion

Five of eight major fatty acids showed statistically significant

differences in this experiment. The predominant differences in the

18:1 and A19:0 contents are not unexpected. As cells proceed from

exponential to stationary phase, decreases in unsaturates with increases

in cyclopropanes have been reported to occur in other bacteria such as

E. coZi, S. marcescens and S. faecalis (21,22,29,31).

Summing up the percentages of the 18:1 and A19:0 fatty acids at

each phase of growth gives the values listed in Table 12. Again these

sums represent the relative amounts of 18:1 fatty acid incorporated into

membrane lipids during growth. The sums decrease approximately 9

percentage points from mid exponential to late stationary phase. Since

A19:0 shows an increase of 10 percentage points and the 18:1 fatty acid

a decrease of 18 percentage points, it becomes apparent that nearly half

of the decrease in the 18:1 content is due to conversion to the 619:0

fatty acid and the other half is due to decreasing amounts of the 18:1

fatty acid being incorporated into membrane lipid. Therefore, it seems

that S. lactis adjusts its membrane fatty acid composition, in response



































Figure 16. Percentages of the 14:0 fatty acid from S. lactis at various
times during growth.





61






60






50






40






30






20






10






0
5 10 15 20 25
Time (hrs)


































Figure 17. Percentages of the iso 16:0 fatty acid from S. Zactis at
various times during growth.






















50 t


40 L


30 L


20 L


10 L


w


Time (hrs)


w-


d


ra-- -- II I



































Figure 18. Percentages of the 16:0 fatty acid from S. lactis at various
times during growth.






65






60







50







40







30

C-
w




20







10







0
5 10 15 20 25

Time (hrs)




































Figure 19. Percentages of the 16:1 fatty acid from S. lactis at various
times during growth.





67






60







50







40







30






20







10







0 0 *
5 10 15 20 25

Time (hrs)



































Figure 20. Percentages of the 18:0 fatty acid from S. lactis at various
times during growth.





69





60






50






40






J 30






20






10





,0 !
0
5 10 15 20 25
Time (hrs)



































Figure 21. Percentages of the 18:1 fatty acid from S. lactis at various
times during growth.












60 I


50 L


40 L


30 r


20 1


10 L


5 10 15 20 25
Time (hrs)


































Figure 22. Percentages of the A19:0 fatty acid from S. Zactis at various
times during growth.




















50 L


5 10 15 20 25
Time (hrs)



































Figure 23. Percentages of the 21:0 fatty acid from S. lactis at various
times during growth.






































































Time (hrs)


60 I


50 .


40 1


10 1


w--


M-


I ~U


~---- -' -1
















Table 11. F-values and
experiment.


significance of differences for growth phase


Fatty Acid F-valuea Significance




14:0 50.17 *

iso 16:0 6.04 *

16:0 1.61 NS

16:1 2.65 NS

18:0 0.53 NS

18:1 132.96 *

A19:0 77.44 *

21:0 15.10


a F =2.77
.*5,5,18
b = significant; NS = not significant
















Table 12. Sums of 18:1 and A19:0 fatty
of growth.


acid percentages at each phase


Phase of growth 18:1 A19:0 18:1 + A19:0





Early exponential 57.4 2.1 59.5

Mid exponential 58.0 3.0 61.0

Late exponential 53.0 5.1 58.1

Early stationary 45.6 8.4 54.0

Mid stationary 42.0 11.7 53.7

Late stationary 39.7 12.1 51.8





78



to changes in growth phase, primarily in two ways it decreases its

overall incorporation of 18:1 fatty acid and increases it content of

619:0 fatty acid.

















CHAPTER IV
EFFECTS OF VARIOUS CARBOHYDRATE SOURCES ON THE
FATTY ACID COMPOSITION OF S. lactis



Literature Review

Reports on the influence of carbohydrate source on bacterial fatty

acid composition are scarce. In 1974, Drucker, Griffith and Melville

(15) compared fatty acid profiles of two strains of S. mutans and one

strain of S. faecalis when grown on 1.0% glucose and 3.0% sucrose.

The iso 14:0 was 35 percentage points higher in one strain of S. mutans

grown on the sucrose compared to growth on the glucose. S.

faecalis showed a similar result, but only 7 percentage points. The

same strain of S. mutans showed lesser amounts of 16:0 and 16:1

fatty acids when grown on sucrose, 20.0% and 4.0% respectively. The

same organism also demonstrated 12 percentage points less 18:1 fatty

acid on sucrose compared to glucose. S. faecalis had 5

percentage points lower 18:0 content and 10 percentage points lower

18:1 content on sucrose compared to glucose. Carbohydrate source

does have an effect on fatty acid composition, but it depends on the

organism as well as the carbohydrate.



Additional Methods

Organisms were grown in 0.056 M concentrations of the following

carbohydrates: trehalose, dextrose, lactose, glucosamine, cellobiose,

fructose, galactose, mannose, salicin, maltose, xylose, ribose, and

mannitol.

79









A doubling time for growth on each carbohydrate was determined from

growth curves. This experiment was run in triplicate.


Results

Doubling times for growth on the different carbohydrates are

listed in Table 13. Growth was most rapid on trehalose, while mannitol

accommodated the slowest growth.

The fatty acid percentages for growth on each carbohydrate are

listed in Table 14. The 18:0 fatty acid content remained fairly

constant. The 14:0 and 16:1 fatty acids showed relatively small

differences (5 percentage points at most). These same fatty acids,

however, did reach their highest levels in cells grown on carbohy-

drates generating the slowest growth. The largest difference in the

16:0 fatty acid content was 9 percentage points with no obvious rela-

tionship to growth rate. The 21:0 fatty acid remained constant, also

with one relatively high percentage value when the organism was grown

on fructose.

Again the 18:1 and A19:0 fatty acids demonstrated the only dramatic

differences. A difference as large as 30 percentage points was seen

between growth on trehalose and xylose in the 18:1 fatty acid with

the least amounts again somewhat related to slow growth. The

A19:0 fatty acid demonstrated its largest difference of about 16

percentage points between growth on fructose and xylose with the

highest amounts related to slow growth. Figures 24-30 graphically

demonstrate these results.

















Table 13. Doubling times of S. lactis grown on various carbohydrate
sources.



Carbohydrate Doubling time (min.)




Trehalose 42

Dextrose 55

Lactose 65

Glucosamine 67

Cellobiose 72

Fructose 75

Galactose 90

Mannose 90

Salicin 90

Maltose 116

Xylose 183

Ribose 342

Mannitol 391



















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