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A COMPARATIVE STUDY OF THE
MORPHOLOGICAL AND PHYSIOLOGICAL
CHARACTERISTICS OF NITRATE REDUCING

AND NON-RECUCING STRAINS 0F

LACTOBACELLUS PLANTARUM

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MICHIGAN STATE UNIVERSITY

DonaId Lewis Robach
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A COKPARATIVE STUDY OF THE IORPHOIOGICAL AND PHYSIOLOGICAL
CHARACTERISTICS OF YITRATE REDUCING AND
NON-REDUCING STRAINS OF

' LACTOBACILLUS PLANTARUM

By

Donald Lewis Robach

AN ABSTRACT

Submitted to the College of Arts and Science
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of
MASTER OF SCIENCE
Department of Microbiology and Public Health

1957

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Approved byjzzécfl/ QM
1 V *

Donald L. Robach
1

The purpose of this study was to determine if there were any

taxonomic differences between strains of Lactobacillus plantarum which

 

reduced nitrate in indole-nitrate medium (BBL) and those which did not.

Five of the 10 cultures used in this investigation reduced nitrate.

All cultures had similar cellular and colonial morphology.
They were all catalase negative, homofermentative, and microaerOphilic.
However, variations were noted in other physiOIOgical studies. One
strain produced abnormally high titratable acidity in skimmed milk, formed
dextro-rotary lactic acid and failed to ferment raffinose and melibiose.

Therefore, it was classified as Lactobacillus casei. Differences between

 

the other 9 strains were noted in the hydrolysis of esculin and sodium
hippurate, nutritional requirements, and acid production from various
carbohydrates. All 9 strains apoeared to fit into the classification as

given in Bergey’s Manual (1948) for L? plantarum with the exception that

 

5 cultures reduced nitrate. However, the variations were such that none

of these cultures could be placed in any one of the three groups proposed

by ROgosalgtigl. (1953) fer strains of E. plantarum. No other taxonomic
characteristics were correlated with nitrate reduction. Therefore, it was
concluded that nitrate reduction should be included as one of the variable
characteristics of this species and that no further taxonomic division of

the cultures should be made.

1. BREED, R, 8,, LURRAY, E, G. D., and KITCHENS, A. P. 1948 Bergey's Manual

of Determinative Bacteriology. 6th. ed. Williams and Wilkins cc.,
Faltimore, Md. .-

 

 

2. ROGOSA, a. 'z‘v'., Z‘IIITCHELL, J. A., TIBETAN, R. F., and DISEALLY, N.,
assisted by BEAKAN, A. J. 1953 Species differentiations of oral
lactobacillus from man including descriptions of Lactobacillus salvarius

 

nov. spec. and Lactobacillus cellobiosus 10v. Spec., J. BacteriEI.,

EST s‘T‘s ~699.

 

A COMPARATIVE STUDY OF THE MORPHOLOGICAL AND PHYSIOLOGICAL
CHARACTERISTICS OF NITRATE REDUCING AND
NON-REDUCING STRAINS OF

LACTOBACILLUS PLANTARUM

By
Donald.LewiI Rebach

A THESIS

Submitted to the College of Arts and Science
Michigan State University of Agriculture and
Applied Science in partial fulfillment of

the requirements for the degree of
MASTER OF SCIENCE
Department of Micrdbiology and Public Health

1957

ACKNOWLEDGEMENT

The author wishes to express his sincere appreciation to
Dr. Ralph N. Costilow, Associate Professor of.Microbiology and
Public Health, for his ever ready and much needed advice,

helpful suggestions and valuable criticisms.

TABLE OF CONTENTS

PAGE
INTRODUCTION............................................................l

REVIEW OF LITERATURE....................................................2
Micrdbiological Reduction of Nitrate..........................2
Classification of L, plantarum................................7

Vitamin and Amino Acid Requirements of L. plantarum...........9
EXPERIMENTAL PROCEDURES AND METHODS.....................................15
Source and Method of Handling Cultures........................15
Morphological Studies.........................................15
Physiological Studies.........................................l6
RESUETS.................................................................l9
Morphology....................................................l9
Physiology....................................................2O
DISCUSSION..............................................................33

LIST OF TABLES

TABLE PACE

Hydrolysis of sodium hippurate and esculin and final
titratable acidity in skimmed milk ...........................2l

The fermenting ability of various strains of L, plantarum
on some carbohydrates in cystine trypticase agar..............22

The fermenting ability of various strains of L. plantarum
on some carbohydrates in Rogosa's medium No. 3................23
and

2h

The effect of the omission of various vitamins from a
synthetic medium on the acid production by various strains
Of L. ElantarIJmOOOOOOOOOOOOO.COO....0OOOOOOOIOOOO0.0.00.00.00.27

The effect of the omission of various amino acids from a

synthetic medium on the acid production by various strains
or L. EhntarmOOOOOOIOOO00.0.00...OOOOOOOOOOOOOOOOOO... 000000 28

The effect of the omission of various amino acids from a
synthetic medium on the acid production by various strains
Of LO ElantarumOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO.IO. ...... 29

Water of crystallization in zinc lactate, and optical
activity of lactic acid.......................................3l

INTRODUCTION

Strains of lactic acid bacteria similar to Lactdbacillus
plantarum have been found to reduce nitrates in indole-nitrate medium
(BBL) (Costilow and Humphreys, 1955; and Costilov gt_gl., 1956). The
authors Observed that about one-half of the strains isolated had this
ability. These isolates were found to be similar in.many other res-
pects to Lactobacillus plantarum but a complete comparative study'was

not made.

It is possible that nitrate reduction by this organism was
not previously noted due to the test medium employed. Sacks and
Barker (l9h9) as well as a number of other investigators have established
that oxygen tension is a very important factor in the ability of bacteria
to reduce nitrates. The composition of the medium.may also influence
the results obtained. Also, some investigators retained only strains
which did not reduce nitrates, discarding all nitrate positive strains

as a preliminary procedure (3.5., see Rogosa gt_al., 1953).

The present study is to determine whether those strains which
have the ability to reduce nitrates should be included in the species
Lactdbacillus plantarum or be designated as a separate species from

organisms which do not reduce nitrates.

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REVIEW OF LITERATURE

Microbiological.Reduction g£_Nitrate

According to Conn (1936), for a number of years one of the
paramount features in the characterization of bacteria has been the
ability to reduce nitrates. In most papers in which species are des-

cribed and in Bergeyjs Manual (l9h8), a statement is given as to

 

whether or not nitrates are reduced. Generally Just a categorical
statement, nitrates reduced or nitrates not reduced, is made as if the
determination was so concise that no specifications of methods and
conditions were necessary. This, however, is not the case for many
factors enter into the determination. A fundamental prerequisite is
to have a suitable medium for the nitrate test; i,g,, the proper
nutrients to support growth. Another basic factor is the Eh of the
medium. It was demonstrated in the classic experiments of Gayon and
Dupetit (1886) that oxygen inhibits the reduction of nitrate and the

formation of nitrogen by denitrifying bacteria.

Weissenberg (1897) using three denitrifying bacteria found
that complete denitrification occurred only in anerdbic cultures,
whereas the nitrate was reduced only as far as nitrite in aerobic

cultures.

Strickland (1931) determined the influence of oxygen at
various partial pressures on the reduction of nitrate to nitrite by

cell suspensions of Escherichia coli. Under conditions of aeration

that should have maintained an equilibrium of oxygen distribution
between the liquid and gas phases, he found as little as 0.36 per

cent oxygen caused a 21 per cent inhibition of nitrate reduction,

1 per cent oxygen caused approximately 50 per cent inhibition, and

3.76 per cent oxygen caused 93 per cent inhibition. A tenfold increase
in nitrate concentration did not modify these results; thus

demonstrating that the inhibition was non-competitive.

Van Olden (19h0) was first to apply modern manometric
techniques to the study of denitrification. Using Micrococcus
denitrificans, he found that the ability of washed bacteria to pro-
duce nitrogen from.nitrate dependent upon their previous history.
Bacteria that had grown anaerdbically with nitrate were capable of
causing rapid denitrification of nitrate under anaerdbic conditions,
whereas bacteria grown aerobically either with or without nitrate
denitrified very slowly or not at all. van Olden concluded that nitrate
reductase is an adaptive enzyme in a sense. However, from his results
it is impossible to decide which enzyme or enzymes failed to develop
under conditions unsuitable for denitrification. Since nitrite was
formed by some of the bacteria grown aerobically, it is possible that
their inability to denitrify was at least partially due to the absence
of nitrite reductase or'some enzyme mediating a reaction between nitrite

and nitrogen.

Lemoigne gt 2;. (l9h6) found that when.Bacillus me atherium,

was grown in a medium containing nitrate as the sole source of nitrogen,

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a pure oxygen atmosphere greatly increased the lag period. If there
was a source of organic nitrogen in the medium or if the atmosphere
contained less than 6h per cent oxygen, no great lag period was ob-
served. From this they concluded that the mechanism involved in the
assimilation of nitrate was arrested by oxygen; a conclusion that seems
to harmonize with the findings of Weissenberg (1897) that the reduction

of nitrite is especially susceptible to the inhibitory action of oxygen.

In general, except for a few workers, the evidence summarized
strongly indicates that oxygen has a deleterious effect on the re-
duction of nitrate and nitrite and that one or more of the enzymes
involved in denitrification is adapative in the sense that it is only

formed by bacteria grown anerobically in the presence of nitrate.

Woods (1938) found that Clostridium welchi and certain
strains of Escherichia coli reduced nitrate to ammonia with the
utilization of four moles of molecular hydrogen. Nitrites and
hydroxylamine were shown to be intermediates in this reaction. Mole-

cular hydrogen was activated by the enzyme hydrogenase.

Lascelles and Still (l9h6) reported that E, 321i reduced
nitrate to ammonia only in the presence of benzyl viologen as a carrier.
Benzyl viologen is a dye which is reversibly reducible. In the ab-
sence of a carrier, the reduction did not proceed beyond the nitrite
stage. This and the action of some inhibitors suggested that different
enzymes are responsible for the reduction of nitrate from those re-

ducing nitrite and hydroxylamine.. Colter and Quastil (1950) have

rJ

I

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ri

shown that hydroxylamine reduction to ammonia and molecular nitrOgen

can be catalyzed by hemoglobin.

Sacks and Barker (1949) found that oxygen affects nitrate
reduction and denitrification in two ways; by supressing the formation
of enzyme systems that catalyze these reactions, and by directly inter-
fering with the action of enzyme systems when they are present in the
bacteria. They concluded that the exposure of bacteria to oxygen
during growth supresses the formation of enzyme systems responsible
for nitrite reduction much more than those responsible for the reduction
of nitrate to nitrite. This was based on the observation that nitrate
could be reduced only as far as nitrite in oxygen tensions of about
5 per cent. At lower oxygen tensions there was an abnormally large
accumulation of nitrite, but this was accompanied by denitrification .
Both the accumulations of nitrite and the rate of denitrification were

greatly affected by relatively small changes in oxygen level.

Krasna and Rittenberg (1954) have shown that both whole

cells and extracts of Proteus vulgaris catalyze the reduction of nitrate

 

to nitrite by molecular hydrogen. They, also, found that no nitrate
could be reduced until all the oxygen was removed; therefore, the
lenth of induction period was dependent on the oxygen content of the

system.

Nason and Evans (1953) working with nitrate reductase of

Neurospora found that it catalyzed the reduction of nitrates to nitrites

 

according to the equation; 1103+ TPNH ' H-éNOzl-k TPN + 320. The enzyme,

which had been concentrated approximately seventy fold, had a pH optimum

at 7.0 and was observed to be a flavo protein with flavinadeninedinucleo-
tide (F. A. D.) as the prosthetic group. It was suggested that sulfhydryl
(-33) groups were present on the enzyme as well as a heavy metal constituent.

It was of an adaptive nature and not stimulated by molybdenum.

Nicholas et a1. (1954) found that cell free extracts of

molybdenum-deficient Neurcspora crassa showed a striking decrease in

 

nitrate reductase (from one-tenth to one-thirtieth of the controls).
Individual deficiencies of other micronutrient elements (salts of iron,
manganese, zinc, magnesium, boron or copper) did not result in a de-

crease of the enzyme. This is in agreement with Nason and Evans (1953).

Nicholas et a1., also, found that both Neurospora crease and Aspergillus

 

 

gigs: required molybdenum when nitrate, nitrite or ammonia was used

as the sole source of nitrOgen. When ammonia was the sole source of
nitrogen, nitrate reductase was not formed and the molybdenum requirement
was less. Therefore, this element is required for metabolic processes

other than the reduction of nitrate.

Costilow and Humphreys (1955), found that about one half of

the strains of Lactobacillus plantarum tested reduced nitrate to nitrite

 

when grown in indole nitrate medium (BBL). These same cultures were un-
able to reduce nitrates in nitrate broth (Difco) or in indole nitrate
medium with the 0.1 per cent agar omitted. The addition of yeast extract
increased the rate of nitrate reduction but the oxygen tension of the
test medium appeared to be the deciding factor as to whether nitrate

was reduced by some strains or not.

Classification g£_LactObacillus plantarum

Tittsler (1952) in giving the "Introduction" to the Symposium
on the Lactic Acid Bacteria stated that this group of organisms attracted
the attention of bacteriologists since the beginning of bacteriology
due to their practical importance to the food and fermentation industries.
Lactobacilli, particulary, have been subjected to much basic research
yet there is a great need for satisfactory means of identifying, differ-
entiating, and characterizing the genera and species in the family
LactObacteriaceae. L, plantarum (Orla-Jensen), typifies the confusion
that has existed as illustrated by the nineteen probable synonyms
listed in the sixth edition of Bergey's M2222}: The probable synonyms
are; Bacillus pabuliacidi, Lactobacillus pabuliacidi, Bacillus cucumeris
fermentati, Lactobacillus cucumeris, Bacillus wortmannii, Bacillus
listeri, Lactobacterium listeri, Lactobacillus listeri, Bacillus naercki,
Bacillus leichmanni, Bacillus beijgrinckii, Lactobacillus beijerinckii,
Bacterium brusae asiaticae, Lactobacillus busaeasiaticus, Bacterium
brassicae, Lactobacillus brassicae, Lactobacillus pentosus,

Lactobacillus arabinosus and Lactdbacillus wortmannii.

Pederson (1952) found that lactic acid bacteria obtained
their energy by partial fermentation of sugars without the utilization
of free oxygen. This necessitates the utilization of large quantities
of sugar to obtain relatively small amounts of energy for growth, which

results in comparatively large amounts of determinable fermentation end

products. Therefore, species within a group acting similarly could be
partially identified by these end products of fenmentation. Homo-
fermentative species, of which.§. plantarum.is a.member, produce lactic
acid as a major end product with small amounts of acetic acid and
carbon dioxide from hexose sugar fermentations. Eqpimolar amounts

of lactic acid and acetic acid are produced from the fermentation of

the pentose sugars.

Snell (1952) stated that L. plantarum will ferment up to
95 per cent of the utilized glucose to lactic acid and the remainder of
the sugar is converted to carbon dioxide, traces of volatile acids,
and cellular protoplasm. All of the most recent evidence indicates

that lactic acid is formed by way of the Embden-Meyerhof scheme.

Pederson (1936) discovered the greatest variation between
strains of Lactobacillus plantarum existed in their ability to ferment
individual sugars, ranging from a definite failure to ferment to a
definite positive fermentation. The majority of the strains formed
acid from glucose, fructose, mannose, galactose, sucrose, maltose,
lactose, raffinose, and salicin, and to a lesser extent sorbitol,
mannitol, dextrin, and glycerol. The majority of the strains did
not ferment rhamnose, starch, and inulin. A few strains had only
slight or no action on arabinose and even less on xylose. The same
range of variation occured even where cultures were Obtained from
identical material. This is in near agreement with what is now given

in Bergeyts Manual (19h8). Similar results were also observed by

Rogosa gt_§l. (1953) and Tittsler gt_gl. (l9h7).

Snell (1952) points out that all living organisms require
a utilizable form of energy, apprOpriate nitrogen and carbon containing
compounds to permit synthesis of the various components of their proto-
plasm, and certain inorganic salts for growth. These nutritional re-
quirements for the lactic acid bacteria are among the most complex
so far studied. He further states that the most important compounds
utilized by lactic acid bacteria as a source of energy are the soluble

carbohydrates.
Vitamin and Amino Acid Requirements of L. plantarum

Due to the advent of micrdbiological assay techniques for
various amino acids and vitamins much progress has been made in the
study of the mutational requirements of bacteria. The lactic acid
bacteria in general, because of their fastidious nutritional requirements
have been subjected to meny'investigations. L, plantarum is one of the
lactic acid organisms which.has been commonly used. This review will

be limited to the requirements of this organism and its synonyms.

Vitamin requirements. Peterson and Peterson (l9h5) in
preparing a comprehensive review of the vitamins and growth factors
required by microorganisms noted that biotin, niacin, pantothenic acid
and riboflavin were the most frequently reported as being essential.

All of these vitamins have been established as essential for_§, plantarum.

lO

Snell and his co-workers (1938, 1939, 191+l) have demonstrated the re-
quirements of L. arabinosus 17-5 for nicotinic acid, pantothenic acid,
and biotin. Cheldelin_e‘_t_ _a__L. (19145) using one strain of lactic acid

bacteria labeled as__L. lantarum, two as L. arabinosus, and two as L.

 

pgntosus demonstrated a requirement for pantothenic acid. Rosen and
Fabian (1953) working with ten isolates of L. plantarum frcn cucumber
fermentations demonstrated that each required biotin, niacin, and
pantothenic acid as did L. arabinosus 17-5. This was substiantiated
by Costilow and Fabian, (1951+) using four strains of L. plantarum
isolated from cucumber fermentations. Kreuger and Bterson, (191+8)
reported similar results for L. pgntosus l2h-2. Rogosa 31; _a_1_. (1953)
demonstrated that all strains of the L. plantarum group tested re-
quired niacin and pantothenic acid. Results as to biotin requirements

were not given.

Snell and Mitchell (191+2) using L. arabinosus 17-5 and
L. mntosus noted that p - sminobenzoic acid was nonessential as did
Snell (19,48, 1950, 1951) using L. plantarum and L. arabinosus. Costilow
and Fabian (1951;) noted that three of four L. plantarum strains tested
required p - aminobenzoia acid. Kreuger and Peterson (l9h8) found
this vitamin to be stimulatory for L. plantaru- and L. arabinosus while
Isbell (19h2) and Lewis (191t2) observed it to be essential for L.
arabinosus. In the concentrated media of Shankman 9_t_ 9.34. (19h?) the

omission of this vitamin had no apparent effect on L. arabinosus 17-5

or L. Entosus .

ll

Snell and Strong (1938) noted riboflavin was not required
by L. pgntosus 12h-2, L. arabinosus l7-5, nor by L. brassicae (801d).
Campbell and Hucker (19%) obtained similar results with cultures
labeled L. arabinosus F-l7-5, Bacillus cucumeris fermentatae L-25,
and two strains of L. plantarum. Rogosa gt a_l. (1953) reported that
some strains of L. plantarum required riboflavin which is in agreement

with Rogosa gt 3L. (191W) and Costilow and Fabian (l95h).

Bohonos 9}; EL. (1941, 191+2) reported that neither L. arabinosus
or_I_.. pgntosus required pyridoxine but was synthesized by them. But,
Shankman 9; 5L. (196) found that both L. arabinosus 17-5 and L. brassicae
(801%) were stimulated by pyridoxine when short incubation periods were
used. No stimulation of L. pentosus was observed. Costilow and
Fabian (1951+) noted that [only one of the four strains of L. plantarum
tested required pyridoxine. Rogosa 3: EL. (1953) observed pyridoxine

to be neither required or stimulatory.

Shankman gt EL (1918) and Baumgarten it. a_1. (19%) noted
neither thiamine or folic acid were essential for_L. arabinosus 17-5.
However, the latter workers have observed folic acid to be stimulatory.
L. pentosus did not require these two vitamins, (Shankman et a1. , l9h3).
Rogosa a EL. (1953) using strains of L. plantarum found neither thiamine

or folic acid were essential or stimulatory.

Snell and Wright (19%) observed that pyridoxine, thiamine,
and riboflavin stimulated the growth of L. arabinosus 17-5 during the first

few hours of incubation.

w.

l2

Amino acid requirements: The findings of various workers
on the amino acid requirements of L, plantarum are still.more con-
flicting than on the essential vitamins. Snell (1952) contends that
the number and the identity of amino acids required by lactic acid
bacteria are highly dependent upon the different levels of vitamins
that are supplied in the medium. He concluded that the conflicting
data concerning amino acid requirements of lactic acid bacteria that
appears in the literature is due primarily to this; and, also, to
contamination of certain commercially available amino acids with other
amino acids. To exemplify this statement he puts forth the following
fact: in.media that is low in biotin, Lactobacillus arabinosus re-
quires aspartic acid but grows without it when large amounts of
biotin is supplied. This suggests that biotin is in some way essential
for synthesis of aspartic acid by the organism. This hypothesis has
been confirmed by Lardy g3 gL. (19h9) and Snell (1951) using tracer
experiments. Holden gg.gL. (1951) and Snell (l9h8, 1951s) have shown

that vitamin B6 may also alter an organismfs need for an amino acid.

Even using the same strain of L, plantarum (L. arabinosus
17-5) investigators have found considerable differences in amino
acid requirements. The number of amino acids found to be required
by this strain during six investigations varied from ten (negated, l9hh)
to five (Dunn, l9h7). In all six of the investigations (Shankman, l9h3;
Kuiken fl". g_l_. 19343; Hegsted, 19M; Lyman g‘g gL. , 191+7; Dunn g gL.

l9h7; Costilow and Fabian, 195k) five amino acids (233,, glutamic acid,

13

valine, leucine, isoleucine, and tryptophane) were noted to be essential.
Cystine was found to be presently essential in all cases except by

Dunn (l9h7), who found it to be stimulatory even in an enriched medium.
Rissen gg_gl. (l9h7) also noted that cystine was stimulatory for L,
arabinosus 17-5. Threonine was Observed to be required in three of the
investigations (Shankman, l9h3; Kuiken gg_gL. 19h3; and Costilow gg_gL.
l95h), and methionine was demonstrated to be essential in three (Shankman,
l9h3; Dunn g§_gl. l9h7; and Hegsted, l9hh). Only Kuiken gg_gl, l9h3
found lysine to be required. Phenylalanine was found to be required

in three investigations (Shankman, 19L3; Kuiken.g3_gl, 19h3; and
Hegsted, l9hh); arginine in two (Hegsted, 19hh; Dunn g3 g}, 19h7);

and tyrosine in two (Shankman, 19h3; and Hegsted, l9hh).

Dunn g_‘l_;_ gL. (191+7) using Leuccnostoc mesenteroids noted
considerable variations between strains as to amino acid requirements.
Requirements varied from fifteen amino acids for one strain to two for
another. They further noted that L, pentosus required glutamic acid,
valine, isoleucine, leucine, and cysteine. L, arabinosus 17-5, however,
did not require cysteine, but required tryptophane and in some instances

methionine and arginine.

valine, leucine, isoleucine, glutamic acid, and phenylalanine
were reported by Kruger and Peterson (l9h8) as being essential for L,
pgntosus l2h-2. In the same report it was noted cystine, threonine, and

alanine stimulated growth.but tryptophane showed no effect upon ommission.

‘1

L_‘V

lh

L-tryptophane, L-leucine, DL-isoleucine, DL-valine, L-glu-
tamic acid, L-cystine, DL-threonine, DL-alanine were found to be
required for growth by four strains of L, plantarum isolated from
cucumber fermentations by Costilow and Fabian (195%). They further
found that three strains required DL-phenylalanine and L-tyrosine and

one strain needed L-arginine for growth.

Lyman 93; 11; (19W) demonstrated that L. arabinosus 17-5

 

required threonine, lysine, and alanine if pyridoxine was omitted but

did not require them when pyridoxine was present. This shows conclusively
that there exists a relationship between the composition of the media

used for testing and the amino acids found to be essential. In this
report, it was noted that if CO2 was not available, arginine, phenyl-
lalanine and tyrosine were also required. However, pyridoxine was not

required when all these amino acids were present.

Stokes and Gunness (l9h3) reported that pyridoxamine would
eliminate the requirements of L, arabinosus l7-5 for lysine, thrennine,
and alanine but pyridoxine would not. HOwever, if the basal.medium
containing pyridoxine was sterilized for thirty minutes at 15 pounds
similar results were obtained as when pyridoxamine was used. These
Observations might account for many of the conflicting results

Obtained in the various investigations.

15

EXPERIMENTAL PROCEDURES AND METHODS

Source and method 2; handling cultures: Ten pure cultures

 

 

were used for this study. Seven were isolated from cucumber fermentations

and typed as Lactobacillus plantarum in our laboratory. One strain

 

(B-227) was obtained from the Northern Utilization Research Branch
(N.U.R.B.). Strain 2h6 was obtained from Dr. C. S. Pederson, Cornell,
Agricultural Experimental Station, Geneva, New York. The tenth strain

was L. arabinosus 17-5.

 

Each culture was streaked out on selective lactobacillus
agar plates and isolated colonies picked. Again frequent transfers
on trypticase sugar agar were made and microscopic examination was
performed to insure purity of culture when good growth was observed.
One inoculated tube of each strain was placed in the refrigerator for

reference and a duplicate of each was used for further study.

Morphological studies: Colonial morphology was observed
on both trypticase sugar agar and selective lactObacillus agar (Rogosa
gt aL., 1951) streaked and poured plates. Cell morphology was observed
by use of the microscope. Motility was determined by observation of
growth in a semisolid media (cystine trypticase agar), and by micro-
scopic examination of a hanging drop slide. The cultures were observed
as to growth on nutrient agar slants, spore production, and gram

staining reaction.

PhysiDIOgical studies: Cultures were inoculated into indole

 

nitrate medium (BBL) to test their ability to reduce nitrates. Obser—
vations were made after 3 and 6 days incubation. Testing procedures

followed were as outlined in the Manual 2£_Methods for Pure Cultugg

 

Study of Bacteria (1946) using sulphanilic acid and alpha-napthylamine

to test for the presence of nitrites.

The cultures were observed for catalase production by the
addition of 3 per cent hydrogen peroxide to trypticase agar stab and

trypticase broth cultures.

I"ermentation studies were conducted on the following sub-
strates at a concentration of 2 per cent; adonitol, arabinose, cello-
biose, dulcitol, glucose, inositol, inulin, lactose, levulose, maltose,
mannitol, mannose, melibiose, melezitose, raffinose, rhamnose, salicin,
sorbitol, sorbose, sucrose, trehaloss, xylose, alpha methyl—d-glucoside,
and alpha methyl-d-mannoside. Galactose was used at a concentration
of 1.4 per cent. Basal media used for the fermentation studies were
cystine trypticase agar (BBL) and liquid medium No. 3 of Rogosa it _a_l_l_.
(1953). The indicator was observed daily and a notation made on the
day it changed color distinctly. After two weeks' incubation, the
pH of the contents of each tube containing the liquid media was

observed.

In addition to observing the indicator of the cystine

trypticase agar, notations were made as to motility and gas formation.

 

1'?

Tubed media No. 5 of Rogosa g£_gL. (1953) was inoculated, plugged with

sterile agar, and Observed for gas formation.-

Tests to determine if sodium hippurate and esculin could

be hydrolyzed by the organisms were performed as outlined by Rogosa

22a. (1953).

Sterile skimmed milk was inoculated and incubated for two
weeks at which time it was observed for curd formation and the titrat-
able acidity determined. Reaction of the cultures on litmus milk

was also noted.

Medium No. 5 of Rogosa £2.2l- (1953) was used as the
production medium for determination of optical activity of lactic
acid produced. Arabinose and xylose were not added for all strains
were homofermentative. The medium was dispersed at the rate of 300ml.
per 500ml. Erlenmeyer flask and 10 grams of calcium carbonate added.
It was autoclaved at 15 pounds pressure for 15 minutes. Flasks
were inoculated with lml. of culture which had been grown in.media
No. 5. After 2 weeks of incubation, volatile acids were distilled
off and the lactic acid extracted with ether and finally zinc lactate
formed. The preparation of zinc lactate and determination of optical
activity were performed according to the methods of Currier 22.2l°

(1933) with.modification from the procedure of Brinn gt_gL. (1952).

Nutritional studies were performed using the synthetic

medium of Sauberlich and Baumann (l9h6). Cultures were grown in micro-

18

inoculum broth, centrifuged, washed, resuspended, and diluted with
sterile saline. One drop of this suspension was used to inoculate

the media for nutritional studies. After an incubation of 3 days

the pH of the contents of each tube was measured and the acid titrated

with sodium hydroxide (about 0.1 N) to pH 7.0.

All cultures were incubated at 30°C. All tests were
completed in duplicate except for that of the optical activity of

lactic acid.

19

RESULTS

Five of the ten strains believed to be LactObacillus plantarum
used in this study had been found to reduce nitrates to nitrites on
BBL indole nitrate medium (Costilow 195%, unpublished data). This
was rechecked at the onset of this study and found to be true. The
cultures were divided into 2 groups on this basis. The strains re-
ducing nitrate (N03,!) were 17-5, L-lO, A-6-h, A-231-3 and A-85-2.
The non-nitrate reducing (N0§-) strains were 3-227, 2h6, A-73-l,
A-l60-l and A-2h2-l. Since this study was undertaken to determine
any other differences between these two groups, the data are presented

in a comparative form.

Morphology

 

Cell morphology: All ten cultures studied were gram positive
rods measuring 3/h to 1‘p in width and ranging from 3 to 5‘p in length.
The cells had typical rounded ends and appeared singly, in short chains,
and small groups. They were further characterized as being non-motile

and without the formation of endospores.

Colonial.morphology: Colonies appeared similar on both
trypticase sugar agar and selective lactObacillus agar. All strains
formed circular colonies having smooth, convex surfaces with entire
edges and were opaque, with a slight off-white color. They differed

only in the rate of growth. Growth on agar slants was very slight.

L“

 

20

Physiology

All organisms were catalase negative and in no case was gas
produced from carbohydrate fermentation. All strains grew on sterile
skimmed milk forming acid and causing coagulation. However, there
were differences in the amount of titratable acidity produced and
the rate of curd formation (table 1). Similar results were observed

from litmus milk.

Hydrolysis; Only strain 17-5 of the nitrate positive organisms

 

had the ability to hydrolyze both sodium hippurate and esculin. Strains
A96-4 and APBS‘Z caused hydrolysis of sodium hippurate and L910
hydrolysed esculin. Strain A—231-3 demonstrated no ability to hydrolyze

either.

Nitrate negative strains which demonstrated hydrolysis of
both sodium hippurate and esculin were A973-1, AplBO-l, and A-242-1.
B-227 caused the hydrolysis of sodium hippurate and 246 hydrolyzed

esculin (table 1).

Carbohydrate fermentations. The results of the studies of

 

carbohydrate fermentations in cystine trypticase agar are presented
in table 2, and those in Rogosa's medium No. 3 in table 3. The

data from the two media were comparable in all but 5 instances. In
medium No. 3, strains A923l-3 and APSS‘Z demonstrated acid production

from dulcitol, strains A973-l and Apl60-l produced acid from alpha-

21

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TABLE 3 continued

Nogfstreina

N03/53treine
pH'a are recorded and those which signify acid production are underlined.

17-5 L-lO A-6-h A-231-3 A-85-2

 

 

 

A-160-1 A-2h2-1

2&6 A-73-1

B-227

 

C) .:|
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E42 &:2

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Selicin

Sorbitol

5.9

6.0 5.9

6.0

5.9

6.0

Sorbose

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kg: EL:

5.9

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I!" III]

25

methyl-d-glucoside and the former fermented alpha methyl-d-mannoside
also. No acid production was observed with these carbohydrates in

cystine trypticase agar.

All strains formed acid from cellobiose, trehalose, glucose,
lactose, levulose, maltose, mannitol, mannose, salicin, sorbitol, and
sucrose. No strain formed acid from adonitol, inositol, inulin or

sorbose.

Ape-4 was the only strain which produced acid from dulcitol
on both basal media. Alpha methyl-d-glucoside was fermented by strains
17-5, A96-4, ApZSl-S and Ap242-1 and strains 17-5, 246, and A9242-1
fermented alpha methyl-d-mannoside on both basal media. Three strains,
Ap6-4, AP231‘3 and A985-2 fermented xylose. A985-2, A973-1, and AP
242-1 produced no acid from arabinose, four strains L-lO, A96-4, AP
231-3, and.A985-2 produced no acid from melezitose and 4 strains,
A985-2, A973-1, A916O-1, and A9242-l produced no acid from rhamnose.
£9 plantarum 3-227 was the only strain which did not fennent melibiose

or raffinose.

From the data observed there is no consistent difference
in the fermenting ability of nitrate positive strains compared to

nitrate negative strains.

Vitamin requirements: The medium used to test for vitamin

 

requirements was the basal medium 1 proposed by Sauberlich and Baumann

(1946) for microbiological assays using L. plantarum 17—5 as the assay

26

organism. The effect of the omission of individual vitamins from
various lots of the medium on the acid production of the 10 cultures
tested is given in table h. All strains demonstrated a requirement

for pantothenate, niacin, and biotin. One strain, L-lO was greatly
stimulated by the presence of riboflavin and strain A-85-2 was affected
to a slight extent. Thiamine, pfaminobenzoic acid and folic acid

were not essential for any of the 10 strains. However, it should be
noted that same organisms require either folic acid or pfaminObenzoic
acid but not both together (Baker gt El: 1955). Pyridoxine was not
required by any of the organisms tested but was somewhat stimulatory

for strain A-l60-l.

Amino Acid Reggirements: The same basal.medium and procedure
used for vitamin requirements were employed for amino acid requirements
(tables 5 and 6). All strains tested required leucine, isoleucine,
valine, cystine, tryptophane, and glutamic acid while none of the
strains demonstrated a requirement for methionine, lysine, histidine,
serine, glycine and proline. However, histidine and serine stimulated
strains A-85-2 and 3-227 and glycine stimulated strains A-73-l and A-
l60-l. Seven strains, L-lO, A-85-2, B-227, 2H6, A-73-l, A—l60-l, and
A-2h2-l were noted to require tyrosine. Strains L-lO, A-85-2, 3-227
and A-2h2-l required phenylalanine and strain A-73-l was somewhat

stimulated by its presence.

Threonine was either required or greatly stimulatory to all

strains tested. Four strains, L-lO, A-6-h, A-23l-3 and B-227 demonstrated

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27

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30

a need for alanine while the other six strains (17-5, A—85-2, 2&6,
A-73-l, and A-2h2-l) were somewhat stimulated when it was added to the
media. Only two strains (3-227 and 2&6) required asparagine while
five strains (A-23l-3, A—85-2, A—73-l, A-léO-l and A-2h2-l) produced
less acid when it was omitted. For arginine, it was noted that four
strains required it for high acid production (A-6-h, A-23l-3, A-85-2,

and B-227).

Optical activity gf_lactic acid produced; The optical activity
of the lactic acid produced by the various strains is given in table 7.
There is a good correlation between the water of crystallization of the
zinc lactate salt and the optical activity of the salts. Optically in-
active salts theoretically should contain three moles of water of
crystallization or 18.8%. Optically active salts contain 2 moles or
12.89% water of crystallization. Optically inactive lactate is derived
from optically inactive lactic acid and active lactate from optically

active lactic acid.

The lactate resulting from the lactic acid production by all
cultures except B-227 was inactive. This is evidenced by both the water
of crystallization and the specific rotation.measurements. Therefore,

these strains produced inactive lactic acid.

From the data observed the lactic acid formed by B-227 is a
mixture of the d and 1 forms with the optically active d-lactic acid in

excess. If the salt was pure d-lactate, the water of crystallization

 

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32

would be less (3.. 2., 12.9% instead of 13.65%) and the specific Optical
rotation greater (8.8 instead of 4.2). It must be remembered that

the specific Optical rotation is given for the lactic acid pg: :3
rather than the salt. The rotation of zino lactate is opposite of

the lactic acid therefore, the salt demonstrated levo rotation on the

polarimeter.

33

DISCUSSION

From the biochemical behavior of strain 3-227 (i.g., its
inability to ferment melibiose and raffinose, the formation of an
extremely high titratable acidity in skimmed milk, and the formation
of dextro-rotary lactic acid) it must be typed as Lactobacillus casei
rather than Lactobacillus plantarum. This is according to the char-
acteristics of these organisms as outlined by Rogosa et_al. (l953),
Tittsler £3 21. (19k?) and Bergeyis Manual (l9h8). Therefore, strain

3-227 will not be considered for further discussion.

The fermenting ability of the other cultures tested was in
close agreement with strain 17-5. variations were Observed on the
following carbon compounds; arabinose, dulcitol, melezitose, rhamnose,
xylose, alpha-methyl-d-glucoside, and alpha-methyl-dflmannoside. Similar
variation between strains of L, plantarum were observed by Rogosa
23 31. (1953), Tittsler st 31. (l9h7), Orla-Jensen (19h3), and Harrison
and Hansen (1950). Similar variations are reported in Bergey's Manual
(l9h8). From the investigation of the fermenting ability of the various
strains no significant difference between nitrate positive and nitrate
negative strains was observed. variations as to sodium hippurate
hydrolysis and esculin hydrolysis were noted among the strains but
they were not specific for nitrate positive or negative strains. Such

variations were also noted by'Rogosa st 31. (1953).

This investigation demonstrated that the well known 17-5 strain

34

required the following vitamins; biotin, niacin and pantothenic acid.
The amino acids required were cystine, glutamic acid, leucine, iso-
leucine, tryptophane and valine. This is in agreement with Hegsted
(19M), Kuiken 5; 2L (191+3), Lyman g; 3;. (191a), and Shankman
(1915). Dunn gt 9.}.- (19h7) found that L. plantarum 17-5 did not re-
quire cystine but he was using a greatly enriched medium. The vitamin
and amino acid requirements of the other 8 strains of L, plantarum
tested were noted to be similar to strain 17-5 in respect to the above
stated vitamins and amino acids. All 9 strains either required or
were very dependent on threonine for rapid acid production. This is
in agreement with Costilow and Fabian (1955) using the same basal
medium. Lyman.g£_al, (19h7) and Stokes gt a1. (l9h3) using different

media have shown threonine to be non-essential for L, plantarum.

Slight variations were noted among the isolates tested in
respect to vitamin requirements. Riboflavin was greatly stimulatory
to one strain and slightly stimulatory to a second strain. Pyridoxine
was found to be slightly stimulatory for one strain. In testing for
amino acid requirements a wider variation was noted among the strains
in respect to tyrosine, phenylalanine, alanine, arginine, and
asparagine. Histidine and serine were stimulatory for one strain.
Previous reports have also been conflicting as to the requirements of
this species for these nutrients.- Since the basal.medium was constant
in these experiments the variations noted must be due to differences

in strains rather than to the test medium. Using the same basal medium,

35

Costilow and Fabian (1955) found similar variations; and Dunn 33 El.
(19%?) and Shankman gt al. (19h?) Obtained similar results with
different media. From the observed nutritional requirements, there is

no significant difference between nitrate positive and nitrate negative

strains.

All the strains of L, plantarum investigated produced a
racemic mixture of lactic acid. This is in agreement with several
other investigators (Rogosa gt al., 1953; Tittsler £2,2l°: l9h7; Orla-

Jensen, l9h3; and Pederson, 1936).

The ability of microorganisms to reduce nitrates is a very
important taxonomic tool. It is used extensively not only for bacterial
taxonomy (Bergey's Manual of Determinative Bacteriology, 19MB), but
also for that of yeasts. In fact Lodder and Kreger-VanRij (1952)
used nitrate assimilation not only to demonstrate species differences
but, also, to differentiate genera. The genus Hansenula, in their
taxonomic breakdown, is separated from the genus Pichia only by nitrate
assimilation; Hansenula has the ability to reduce nitrate and Pichia

does not. Since certain strains of Lactobacillus plantarum have been

 

shown to reduce nitrates in indole nitrate medium (BBL), it would
seem logical to use this physiological ability as a means of taxonomic
separation. Rogosa 33 al. (1953) and Tittsler gt al. (l9h7) have
divided strains of E. plantarum into three species, L, plantarum,

Lactobacillus pentosus, and Lactobacillus arabinosus, based primarily

 

36

on the organisms' ability to ferment various carbohydrates. However,
the results obtained in this investigation failed to provide additional
support for such a division. Thus, according to the scheme based on
carbohydrate fermentation as outlined by Rogosa gt_2L, (l953), strains
A-23l-3 and A-85-2 which did not ferment dulcitol in cystine trypti-
case agar could be placed in the species L, plantarum. Conversely,

in medium No. 3 both strains fermented dulcitol; and, therefore,

could not be placed in this species. Using either basal medium, 5
strains (L-lO, 2H6, A-73-l, A-l60-l and A-2h2-l) could be classified
as L, plantarum and one strain (17-5) as L, arabinosus. Strains L-lO
and l7-5 reduce nitrates while the other h strains listed do not. No
strain could be typed as L, pgntosus. Strain A-6-h could not be typed

according to this scheme.

A more extensive study was performed by Rogosa g§_aL. (l953)
whereby they separated L, plantarum into 3 groups. The division was
based on carbohydrate fermentations, the hydrolysis of sodium hippunate
and esculin, and a nutritional pattern. From the results obtained
in the author's work not a single strain would satisfy all of the
requirements of any one of the three groups. However, this does not
mean that these cultures are not L, plantarum. It does mean that
there is grave doubt as to the advisability of dividing this group

of organisms into three separate species.

From the foregoing information, it is apparent that there

are no other common taxonomic characteristics that are correlated with

37

the ability of the strains tested to reduce nitrates. Therefore,

it does not appear advisable to differentiate those cultures which
reduce nitrates as a separate species. There is no more reason to
expect all strains of a species to reduce nitrates than there is to
expect them all to ferment a particular carbohydrate or require a
specific vitamin. There are so many of these factors which vary within
this group of organisms, that it would be necessary to create many
species to satisfy all of the patterns possible. Therefore, it is the
authors' opinion that such variations should be listed as strain

differences rather than species differences for this particular group.

36

SUMMARY

Five of the ten strains typed as Lactobacillus plantarum
used in this study were shown to reduce nitrates in indole nitrate
medium (BBL). The purpose of this work was to determine if there
were any other taxonomic differences between those strains which were
capable of reducing nitrates and those strains which were devoid of

this physiological ability.

The cultures were studied for morphological, cultural
and physiological characteristics. All strains were uniform in that
they were gram positive, non-spore forming, non-motile, rods and
were catalase negative and homofermentative. variations among the
individual strains were noted in the hydrolysis of esculin and
sodium hippurate, fermentation of various carbohydrates, and nutritional
requirements. All but one of the ten cultures produced Optically
inactive lactic acid. The one culture producing the active form was

proven to be Lactobacillus casei.

The variations noted among the nine strains classified
as L, plantarum were in no way correlated with nitrate reduction.
Therefore, it is concluded that nitrate reduction in such instances

should be used only for strain differentiation.

10.

ll.

12.

39

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Date Due

 

 

 

 

 

 

 

 

 

 

 

MAHJI ’59

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Illllllv‘l
1'1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Thesis

Robach, Donald L.
A comparative study of

the ... strains of Lacto-
bacillus Plantsrum.

APR 1 O '58

 

 

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