THE ISOLATION AND ABSORPTION SPECTRUM MAXIMA OF

BACTERIAL CAROTENOID PIGMENTS

BEN SOBIN AND GRANT L. STAHLY
Department of Bacteriology, Ohio State University, Columbus
Received for publication November 13, 1941
The production of pigments by bacteria has attracted attention from the begin-
ning of the study of bacteriology, possibly because the presence of pigment is
one of the characteristics of microorganisms which is most readily observed.
In recent years special emphasis has been placed on the chemical nature of the
pigments and on their probable r6les in the metabolism of bacterial cells.
The bacterial pigments whose chemical structures have been determined fall
into four main groups (White, 1939). Prodigiosin, the red pigment of Serratia
marcescens, is a pyrrole derivative. Pyocyanin, the dark blue pigment of
Pseudomonas aeruginosa, was the first phenazine derivative to be found in nature.
Phthiocol, isolated from the tubercle bacillus, is a naphthaquinone derivative.
However, the largest group of bacterial pigments belongs to the class of com-
pounds known as carotenoids. Most yellow, orange, and red pigmented bac-
teria contain carotenoids. This interesting group of compounds occurs exten-
sively throughout both the plant and the animal kingdoms. In the plant world
the carotenoids are present in forms ranging from bacteria to the highest plants.
Similarly, in the animal kingdom we find these compounds in many forms of
both invertebrates and vertebrates.
Research on the carotenoids in plants and animals has resulted in the develop-
ment of techniques whereby these compounds can be separated and identified.
Because of the ease of obtaining large amounts of material, considerable knowl-
edge of the carotenoids in plants has been gained as evidenced by such mono-
graphs as those of Palmer (1922) and Strain (1938). The investigation of the

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carotenoids present in bacteria has lagged, possibly, because of the greater
difficulties of obtaining sufficient material.
The presence of carotenoids in bacteria was demonstrated by Zopf (1889)
who designated this group of bacterial pigments, llpochromes, because of their
solubility in the fat solvents. Later, Kligler (1914) and Krainsky (1914)
demonstrated the widespread presence of carotenoid pigments in different species
of bacteria.
Only a few of the large number of bacteria known to contain carotenoids have
been subjected to pigment identification. Reader (1925) found beta-carotene
and lycopene in Sarcina aurantiaca and a hitherto unreported pigment, which
she named coralin, in Streptothrix corallinus. Chargaff and Dieryck (1932)
reported the presence of a xanthophyll and a hydrocarbon, which they called
sarcinene, in Sarcina lutea; Nakamura (1936) found a xanthophyll ester in this
bacterium. Chargaff (1933) found zeaxanthin to be the only pigment in Staphy-
lococcus aureus and differed with Reader (1925) in reporting beta-carotene and
265
266 BEN SOBIN AND GRANT L. STAHLY

zeaxanthin in Sarcina aurantiaca. Chargaff (1933) isolated beta- and gamma-

carotenes and Ingraham and Steenbock (1935) isolated alpha- and beta-
carotenes, cryptoxanthin, and esters of lutein, zeaxanthin, and azafrin from
Mycobacterium phlei. Two additional acid-fast bacteria were studied by
Chargaff and Lederer (1935). Bacillus lombardo-pelligrini contained beta-
and gamma-carotenes and the bacillus of Grassberger was found to contain beta-
and gamma-carotenes, and lycopene.
The carotenoid pigments in some purple sulphur bacteria were investigated
by Karrer and Solmssen (1936). Rhodoviolascin, rhodopin, rhodopurpurin,
flavorhodin, and rhodovibrin were isolated and described briefly. Probably
most of these pigments are limited in their occurrence to the sulphur bacteria.
A carotenoid pigment named spirillo-xanthin was isolated from Spirillum
rubrum by van Niel and Smith (1935). Leprotin, a carotinoid hydrocarbon, was
found by Grundmann and Takeda (1937) in an acid-fast bacterium isolated from
a leprous lesion.
The carotenoid pigments most commonly found in bacteria belong to the
following groups: (1) hydrocarbons, such as beta-carotene; (2) alcohols, such as
xanthophyll; (3) esters; and (4) carotenoid acids.
It is clear that more information is desirable concerning the chemical nature of
the bacterial carotenoid pigments. Such knowledge would be an aid in the
solution of fundamental problems, such as the role of these pigments in bacterial
metabolism. Although the structure and certain other characteristics of some
of these pigments have been established, data on other pigments are lacking.
The isolation and spectrometric analysis of the carotenoids are basic preliminary
steps in their investigation. An aspect of the present study has been the adapta-
tion of the methods used for the separation and identification of the carotenoid
pigments of higher plants to the bacterial carotenoids. Absorption spectrum
maxima have been determined and compared with those recorded in the litera-
ture for similar pigments. Complete identification of the pigments which were

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isolated is not claimed but pertinent data are presented.
MATERIALS AND METHODS
Cultivation of the bacteria. The pigmented bacteria were grown on nutrient
agar containing two per cent glycerol and adjusted to pH 7.2. Inoculations
were made by spraying the surface of the medium with a twenty-four-hour
broth culture, using an atomizer through which sterile air was passed. Sixteen-
ounce French square bottles were used for culturing the bacteria. After in-
cubation for one week at room temperature, the cells were removed by the ad-
dition of a 70 per cent aqueous solution of acetone and the scraping of the surface
with a glass rod bent at a right angle. Water was not used because it frequently
forms a hydrophilic suspension with the cells from which the latter are removed
with difficulty. The pigments were not extracted by the aqueous acetone and
the bacterial cells were separated by centrifugation.
Extraction of pigments. Kuhn and Brockmann (1932) employed absolute
methanol for extracting the carotenoid pigments from finely divided plant
 ISOLATION OF BACTERIAL CAROTENOID PIGMENTS 267
material. The present investigators found that cold methanol was effective
in extracting the carotenoids from moist bacterial cells when the mixture was
ground with an abrasive such as alundum. A convenient method of extraction
involved the addition of 50 ml. of methanol to approximately one gram of moist
bacterial cells and the placing of the container in hot water to bring the methanol
to the boiling point quickly. The pigments were extracted in a few minutes.
The solution was then cooled and the cells removed by centrifugation. The
short exposure to an increased temperature was found to produce no injurious
effects on the pigments.
Separation of types of pigments. The methanol extract then was subjected to
partition between immiscible solvents to separate the various types of carot-
enoid pigment. A modification of the method of Kuhn and Brockmann (1932)
is represented schematically in figure 1.
Add water to the methanol extract to give an alcohol
concentration of 90 per cent. Shake in separatory funnel
with petroleum ether.

Petroleum ether phase Methanol phase

(Contains hydrocarbons and (Contains alcohols and
esters.) Evaporate to dryness. carotenoid acids.) Make
Saponify with alcoholic po- alkaline, dilute with water,
tassium hydroxide. Add water and shake with diethyl
to make alcohol concentration ether.
90 percent. Shake with pe-
troleum ether.

Petroleum ether Methanol Ether Aqueous

phase phase phase phase
(Contains hydro- (Contains (Contains Acidify and ex-
carbons.) freed alcohols.) alcohols.) tract free carot-
enoid acids with
diethyl ether.

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FIG. 1. SCHEME OF SEPARATION OF BACTERIAL CAROTENOID PIGMENTS

The methanol extract was diluted with water to give an alcohol concentration
of 90 per cent and then was shaken in a small separatory funnel with an equal
volume of petroleum ether. The petroleum ether layer was drawn off and shaken
several times with fresh portions of ninety per cent methanol. Similarly, the
alcohol layer was shaken repeatedly with petroleum ether in order to insure a
complete separation of the pigments.
The petroleum ether layer was freed from water by shaking with saturated
salt solution and standing over anhydrous sodium sulphate for half an hour,
and then was evaporated to dryness under partial vacuum. The dried pigment
may contain hydrocarbons and esters. Fifty milliliters of a two-and-one-half-
per cent solution of potassium hydroxide in methanol was added and the solution
was kept at 40°C. for three hours. Water was then added to give an alcohol
concentration of 90 per cent and the solution was shaken in a separatory funnel
with petroleum ether.
268 BEN SOBIN AND GRANT L. STAHLY

The petroleum ether layer was drawn off, washed repeatedly, freed from water,
and evaporated to dryness under reduced pressure. The residue was taken up in
petroleum ether and the individual hydrocarbons were then separated by chroma-
tographic analysis.
The freed carotenols in the alkaline methanol layer were forced into diethyl
ether by the addition of half-saturated salt solution. The ether layer was washed
with water until the washings were no longer alkaline to phenolphthalein, freed
from water, and then evaporated to dryness under vacuum.
The original methanol phase containing carotenols and carotenoid acids was
made alkaline to litmus, diluted with water, and shaken with diethyl ether.
The ether portion was washed, freed from water, and evaporated to dryness.
If there are any carotenoid acids present, they remain in the aqueous phase in
the form of their salts. The acids may be recovered by the addition of dilute
hydrochloric acid and extraction with diethyl ether.
The procedures just described cover all four of the main types of carotenoid
pigments; certain steps are eliminated if partition tests reveal the absence of any
of these groups.
Chromatographic adsorption. After separation of the pigments into groups,
the individual components in each group were separated by the chromatographic
adsorption technic devised by Tswett (1906) and excellently described by Cook
(1936). Zechmeister and Cholnoky (1937) have also presented a thorough
description of chromatographic adsorption.
Successful separation of the carotenoids is dependent on the selection of the
proper adsorbent and solvent and on the careful preparation of the adsorption
column.
The choice of adsorbent and solvent depends largely upon the particular group
of carotenoids to be separated. The hydrocarbons have a weak affinity for
adsorbing substances; hence, a strongly adsorbing material must be employed.
Strain (1938) recommended for the separation of leaf xanthophylls a mixture of

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a specially prepared magnesia' with an equal amount of heat-treated siliceous
earth2. Bacterial carotenols were too strongly adsorbed on this mixture but it
proved to be entirely satisfactory for the separation of the carotenoid hydrocar-
bons, when either petroleum ether or ethylene chloride was employed as the solvent.
Calcium carbonate, activated by heating at 1500C. for five hours, followed by
cooling in a vacuum desiccator, served well for the separation of the carotenoid
alcohols. Petroleum ether and carbon disulphide are the solvents of choice for
the carotenols.
Calcium hydroxide and a mixture of aluminum oxide and siliceous earth were
tested as absorbents but were found to be less satisfactory than those described.
The adsorption column must be prepared carefully in order to insure satis-
factory separation of the pigments. The adsorption device used in this investi-
I Micron Brand, magnesium oxide No. 2641, California Chemical Company, Newark,
California.
2 Hyflo super cel F.A. 501, Johns-Manville Co., New York.
 ISOLATION OF BACTERIAL CAROTENOID PIGMENTS 269
gation consisted of a glass tube, 15 cm. long and 15 mm. in diameter, sealed at one
end to a tube of 6 mm. bore and approximately 8 cm. in length. The tube was
supported in a vertical position by attachment to a vacuum flask. A wad of
cotton was placed just above the constricted portion of the tube. The adsorbent
was added in small portions, each of which was packed with a rod to which a
metal disk, slightly smaller than the diameter of the tube, was attached. One
of the solvents was poured onto the column and suction was used to test for the
presence of cracks in the column.
Certain precautionary measures must be observed in chromatographic ad-
sorption. The pigments must be free of water before they are dissolved in the
adsorbing solvent. Small amounts of moisture may be removed by adding
benzene and evaporating in vacuo at 50°C. The presence of a trace of ethanol
also interferes markedly with the adsorption.
The pigment mixture was dissolved in about 10 ml. of solvent and the solution
then was poured onto the adsorption column so that the latter was covered
quickly. As the solution passed through the column the pigments formed a
narrow band near the top. The column then was washed with fresh portions of
pure solvent which caused the adsorbed pigments to move slowly through the
adsorbent. If filtration is slow, gentle suction may be applied. The pigments
are separated gradually into a series of bands each of which represents a distinct
pigment. When several zones were present, continued washing with the solvent
washed through those which were adsorbed weakly. The latter were collected
separately in the suction flask. The zones remaining in the column were sep-
arated mechanically and eluted with ethanol and then filtered from the adsor-
bent, using a mat of siliceous earth.
In addition to its use for the separation of similar pigments, chromatographic
adsorption is useful for pigment identification. If a solution of an unknown
pigment is mixed with that of a known pigment and adsorbed on a column, the
formation of a single colored zone indicates that the two pigments are very similar
or identical. The formation of two zones proves that the two pigments are not

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identical. Similarly, if two pigments found to possess identical or nearly identi-
cal absorption maxima by spectrometric analysis are suspected of being the same
pigment, they can be tested by mixing and adsorbing them on a column. The
formation of a single band indicates their unity; the formation of two zones
proves their dissimilarity.
Spectrometric analysis. The carotenoid pigments are distinguished con-
veniently by their absorption spectra. There are several instruments available
for obtaining such spectra. In this investigation a Bausch and Lomb spectro-
meter, equipped with a constant deviation prism of the Pellin-Broca type, was
used. At a sufficiently low concentration of pigment, the bands of maximum
absorption are symmetrical enough to allow one to determine the mean value of
their boundaries by placing the crossed hairs of the instrument over the darkest
portion of the band. The value so obtained was recorded as the absorption
maximum for each band.
270 BEN SOBIN AND GRANT L. STAHLY

The absorption spectrum maxima of the carotenoids were determined in 95

per cent ethanol, in carbon disulphide, and in chloroform.
A Baly tube was used for varying the amount of solution through which the
light passed. A photographic flood lamp was used as the light source. The
spectrometer was calibrated with the sodiuim D line and checked periodically
with this standard. The absorption maxima obtained were compared with
those recorded in the literature for various carotenoids dissolved in the same
solvents.
TABLE 1
Bacterial cultures used
NAME CULTUIM SO
NUMBER

Flavobacterium arborescens ....... 435 American Type Culture Collection

F. suaveolens .................. 958 American Type Culture Collection
F. esteroaromaticum ........... Cornell University
F. sulphureum ................ 42.70 Ohio State University
F. fecale ...................... Cornell University
Sarcina lutea .................... 8.40 Ohio State University
S. flava ....................... 147 American Type Culture Collection
S. aurantiaca ................. 146 American Type Culture Collection
Micrococcus luteus ............... 379 American Type Culture Collection
M. flavus ...4...................400 American Type Culture Collection
Erwinia lathyri ................. National Type Culture Collection, England
E. ananas ..................... Malaya-Dr. Perry Elrod
Bacterium mycoides .............. 35.10 Ohio State University
Cellulomonas flavigena ........... 482 American Type Culture Collection
Staphylococcus aureus ........... 209 U.S.D.A. Phenol Coefficient Test Strain
Staphylococcus aureus ........... 610
Staphylococcus aureus ........... 614
Staphylococcus aureus ........... 615
Staphylococcus aureus ........... 616 Food poisoning-Ohio State University
Staphylococcus aureus ........... 617

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Staphylococcus aureus ........... 620
Staphylococcus aureus ........... 626
Staphylococcus aureus ........... 628 Throat infection-Ohio State University
Staphylococcus aureus ........... 628a Animal infection-Ohio State University
Staphylococcus aureus ........... 628b Furuncle-Ohio State University
Staphylococcus aureus ........... 628c

Bacterial cultures. The pigmented bacteria used in this study are listed in
table 1.
EXPERIMENTAL
The absorption maxma, as determined by spectrometric analysis, for 12
different pigments are recorded in table 2. The pigments were isolated from 14
different species of bacteria and consist of 7 carotenols and 5 hydrocarbons. No
esters or carotenoid acids were isolated from these bacteria. The number of the
pigments, as referred to in table 2, is arbitrary and is used to identify the several
pigments.
 ISOLATION OF BACTERIAL CAROTENOID PIGMENTS 271

Flavobacterium. Inspection of table 2 shows that the single carotenol pig-

ments isolated from F. esteroaromaticum, F. suaveolens, and F. fecale had the same
absorption spectrum maxima. Additional evidence for their identity was ob-
tained by subjecting a mixture of the three pigments to chromatographic
adsorption on a column of calcium carbonate. A single pigmented zone was
obtained. A search of the literature failed to reveal any carotenol possessing the
same absorption maxima as the pigment isolated from these three species of
Flavobacterium.
TABLE 2.
Absorption maxima in different solvents of pigments isolated from various bacteria.
ABSORPTION MAIMA (MU)
BACTERIUM
BACTE
TYPE OF
CARTNM
ABOTENOID PIG-OF COLOR
NO.
MENT
AND ZONE
IN ADSORPTION
COLUMN _______________
CS2 CHC13 C2H6OH

Flavobacterium esteroaromaticum.. Alcohol 1 Orange 453 482 513 460 488 452 482
F. suaveol en .Alcohol 1 Orange 453 483 514 459 488 451 482
F. fecale .Alcohol 1 Orange 453 482 513 459 488 451 483
F. sulphureum .Hydrocarbon 2 Yellow 437 466 499 451 481 440 470
Hydrocarbon 3 Zone 1-orange 465 498 536 450 480 516 440 470 503
Hydrocarbon 4 Zone 2-red 495 530 572 477 508 546 463 494 530
F. arborescens .Hydrocarbon 5 Zone 3-orange 465 500 540 450 480 515 439 469 503
Hydrocarbon 2 Zone 4-yellow 435 465 499 450 480 439 469
Hydrocarbon 6 Zone 5-orange 465 500 540 450 480 515 439 469 503

a
c
Alcohol 7 Zone 1-yellow 460 496 451 480 439 470
Alcohol 8 Zone 2-yellow 464 499 450 480 440 470
S. flaca .Alcohol 7 Yellow 460 495 450 480 440 470
S. aurantiaca.Alcohol 9 Orange 479 514 463 498 456 487

M
f Alcohol 7 Zone 1-yellow 460 495 451 480 440 470
Alcohol 8 Zone 2-yellow 465 499 450 480 440 470
M. flav ........................ Alcohol 7 Yellow 459 494 449 480 439 469
Erwinic lathyri ................... Alcohol 10 Yellow 478 513 458 485 452 483
E. ananas ........................ Alcohol 11 Yellow 474 508 460 493 450 480
Bacterium mpcoides ................ Alcohol 12 Red 477 508 548 454 487 521 446 474 506

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Cellulomoaigena..... Alcohol 7 Zone 1-yellow 460 496 451 480 440 470
1 Alcohol 8 Zone 2-yellow 464 499 450 480 440 470

A single carotenoid hydrocarbon was obtained from F. sulphureum. A

strongly adsorbed yellow zone was obtained with the specially prepared mag-
nesium oxide mixture. Chargaff and Dieryck (1932) obtained a hydrocarbon
from Sarcina lutea which had the same absorption maxima when dissolved in
petroleum ether as has this pigment. They named their pigment sarcinene.
Whether the two pigments are identical is not certain since none of Chargaff's
pigment was available for chromatographic analysis.
The delicacy of chromatographic adsorption is exemplified by the separation
of the pigments of F. arborescens. Partition experiments showed the presence
of hydrocarbons only. Five distinct zones were obtained on the magnesium
oxide mixture when ethylene chloride was used as the solvent. The third and
fifth zones had identical absorption maxima in the three solvents. The orange
272 BEN SOBIN AND GRANT L. STAHLY

pigment from the first zone was also very similar. The three zones, however,
were separated from each other on the adsorption column by distinctly different
pigments. When these three pigments were mixed and adsorbed on a column of
magnesium oxide, three zones were obtained, thus indicating their individuality.
Molisch (1914) obtained a pigment from a sulphur bacterium which he called
alpha-bacteriopurpurin. 'He determined its absorption maxima in carbon
disulphide only; the values were the same as those obtained for the red pigment
from zone 2. Perhaps, the yellow pigment from the fourth zone is the sarcinene
of Chargaff and Dieryck.
When F. arborescens was grown on a medium containing glycerol or glucose,
a red pigmentation was obtained; when grown on nutrient agar, an orange
growth was produced. Pigment analysis, however, indicated only a quantita-
tive difference; the same five hydrocarbons were identified. The amount of the
red pigment was increased by growth on media containing glucose or glycerol.
Sarcina. Two carotenoid alcohols were obtained from S. lutea. As noted
before, Chargaff and Dieryck reported a hydrocarbon which they called sar-
cinene in this species. A second strain of S. lutea was investigated in this labora-
tory and yielded the same two carotenols but no hydrocarbon. It is interesting
to note (table 2) that the absorption maxima were almost identical in chloroform
and in ethanol. When the two pigments were mixed and readsorbed on calcium
carbonate, two zones were obtained again. Pigments with these absorption
maxima have not been reported previously.
S. flava contained a single carotenol which apparently was identical with one
of the pigments of S. lutea as indicated by absorption maxima and adsorption of
the mixed pigments.
S. aurantiaca contained a single carotenol which was unreported previously.
Micrococcus. Two carotenols were found in M. luteus. Absorption maxima
and chromatographic adsorption with mixed pigments indicated that these were
the same as the alcohols obtained from Sarcina lutea. The two species of bacteria

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were differentiated, however, by their fermentation reactions.
A single carotenol was isolated from M. flavus. It apparently was the same
pigment as one of those found in S. lutea, S. flava, and M. luteus.
Erwinia. Single and distinct carotenoid alcohols, both unreported previously,
were isolated from E. lathyri and E. ananas.
Bacterium mycoides. This organism is not to be confused with Bacillus my-
coides. An intense red pigmentation was obtained which was due to a single
red carotenol. A pigment, isolated by Karrer and Solmssen (1935) from a
purple sulphur bacterium and named by them rhodopin exhibited the same
absorption maxima.
Cellulomonas flavigena. This bacterium produced two carotenols. Absorp-
tion maxima and mixed chromatographic adsorption indicated that these pig-
ments were identical with those obtained from S. lutea and M. luteus.
Staphylococcus aureus. Twelve strains of S. aureus obtained from various
sources and with different histories were subjected to pigment analysis. Table 3
presents pertinent data. All the strains examined were found to contain a
 ISOLATION OF BACTERIAL CAROTENOID PIGMENTS2273
hydrocarbon whose adsorption maxima coincided with delta-carotene reported by
Winterstein (1933) to occur in the fruit hulls of Gonocaryum pyriforme, and also
a carotenoid alcohol whose absorption maxima were identical with rubixanthin
isolated by Kuhn and Grundman (1934) from rose hips. In addition to these
two pigments, four strains isolated from food poisoning outbreaks and four
strains from staphylococcic infections contained an ester of rubixanthin.
The strain of S. aureus used by the Food and Drug Administration of the U. S.
Department of Agriculture as the test organism for determining phenol co-
efficients contained, as a third pigment, a hydrocarbon with absorption maxima
identical with those of sarcinene. Chargaff (1933) reported that zeaxanthin
TABLE 3
Pigments isolated from various strains of Staphylococcus aureus

STRAIN ;OulllCE
PIGMENTS
Hydrocarbon I Alcohol Third pigment
209 Food and Drug Ad- *Delta-carotene tRubixanthin tHydrocarbon-sarcinene
ministration
614 Food poisoning Delta-carotene Rubixanthin tEster of rubixanthin
615 Food poisoning Delta-carotene Rubixanthin Ester of rubixanthin
616 Food poisoning Delta-carotene Rubixanthin
617 Food poisoning Delta-carotene Rubixanthin
620 Food poisoning Delta-carotene Rubixanthin
626 Food poisoning Delta-carotene Rubixanthin tEster of rubixanthin
628 Infection Delta-carotene Rubixanthin Ester of rubixanthin
628a Infection Delta-carotene Rubixanthin Ester of rubixanthin
628b Infection Delta-carotene Rubixanthin Ester of rubixanthin
628c Infection Delta-carotene Rubixanthin Ester of rubixanthin
* This pigment had absorption maxima identical with those of delta-carotene.
t Same absorption maxima as for rubixanthin.

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$ Same absorption maxima as for Chargaff and Dieryck's sarcinene.

was the only pigment in S. aureus. In none of the twelve strains studied was
this pigment isolated.
DISCUSSION
It has been an important feature of this investigation to adapt some of the
methods used for the separation and identification of the carotenoid pigments
found in plant material to the separation and the determination of absorption
spectrum maxima of the carotenoid pigments present in bacteria. It was found
possible to make with ease a pigment analysis starting with approximately one
gram of moist cells.
One of the problems encountered in this study was the extraction of the
carotenoids from the bacteria. The extraction of carotenoids from plant ma-
terial may be accomplished by the use of any one of many of the fat solvents.
This was not true for the bacterial carotenoids. It was discovered that the
274 BEN SOBIN AND GRANT L. STAHLY

extraction of the carotenoids from the bacterial cells could be accomplished best
in the presence of a small amount of water with a water-miscible fat solvent.
Methyl alcohol proved to be well suited for this purpose.
Formerly, when carotenoid pigments were isolated they were given names,
often without regard to their chemical structures. Sometimes, the name
signified the source, e.g. sarcinene from Sarcing lutea and violacein from Chro-
mobacterium violaceum. More recently, as new pigments have been isolated,
names have commonly been withheld until the chemical structure of the pig-
ments could be determined. This appears to be a rational policy, especially
since source names lose their significance when the same pigment is found in
different kinds of plants or animals.
The present investigation resulted in the isolation of several carotenoid pig-
ments with absorption maima previously unreported. Some bacteria were
found to contain but one pigment while others contained several. With the
exception of those in S. aureus, the carotenoid pigments in any one species of
bacteria belonged to a single type, with the alcohols predominating. No
carotenoid acids were found in any bacterium.
Several of the different species of bacteria contained identical pigments. An
interesting problem arises as to whether pigment analysis should be included in
securing characteristics for differentiation of bacteria. Consistency of pigment
production is important in this respect. It has been found during the course
of this study that each bacterium produced the same pigments repeatedly.
When F. arborescens was grown on a medium containing glycerol or glucose, a
red pigmentation resulted; on nutrient agar, an orange growth was obtained.
However, upon pigment analysis the same five hydrocarbons were obtained in
each instance with the amount of red pigment enhanced when the organism was
grown on glycerol or glucose. Data are too few, however, to conclude that media
have no effect on the kinds of pigment formed by a bacterium.
It is probable that different strains of a certain species of bacterium will be

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found to produce different pigments. Reader (1925), Chargaff (1933) and the
present authors disagree on the pigment formed by S. aurantiaca. Likewise,
Nakamura (1936), Chargaff and Dieryck (1932) and the present authors fail to
agree with respect to the pigments of S. lutea. In order to test the hypothesis that
different strains of one species produce different pigments when grown on a
standard medium, the present investigators subjected twelve strains of S. aureus
to pigment analysis. Two pigments were common to all, with some variation
in regard to a third pigment. Again these results do not coincide with those
of Chargaff (1933). Insufficient data on the subject are available to attempt
a correlation of pigment type with other properties or with source.
There are interesting opportunities for research in determining the effects of
environmental factors on the kinds and amounts of the carotenoid pigments
produced by bacteria. Rapid growth makes possible a large number of studies
in a short time, the environment may be controlled fairly well, and complicating
factors are fewer, in general, than they are for the higher plants. For the above
reasons, it appears also that bacteria offer definite possibilities in connection with
 ISOLATION OF BACTERIAL CAROTENOID PIGMENTS 275
discovering the true function, if any, of the carotenoids in plants and animals.
Two principal types of reactions have been attributed to the yellow pigments in
leaves. One involves chemical reactions taking place in photosynthesis; the
other is concerned with various oxidation-reduction reactions in which hydrogen
is transferred, presumably by the pigments acting reversibly as hydrogen ac-
ceptors and donors. However, careful consideration of the evidence leads to the
conclusion that the subject is still in need of much critical investigation.
It is known that some naturally occurring pigments, such as riboflavin,
cytochrome, pyocyanine, phthiocol and toxoflavin function as hydrogen trans-
porters in bacterial metabolism. It is evident that studies should be extended to
investigations of the functional role of the carotenoids in bacteria with the ulti-
mate purpose of discovering their role in plants and animals in general.
SUMMARY
Methods for the extraction, isolation, and determination of absorption
spectrum maxima of bacterial carotenoid pigments have been devised or adapted
from those applicable to the higher plant pigments. The pigments were ex-
tracted from bacterial ceIls with hot methanol. They were separated by
chromatographic adsorption and then subjection to spectrometric analysis.
Twelve carotenoid pigments were isolated from 14 different species of bacteria.
These included five species of Flavobacterium, three of Sarcina, two of Micro-
coccus, two of Erwinia, one of Bacterium, and one of Cellulomonas. Seven of
the carotenoids were alcohols and five were hydrocarbons. Some bacteria
produced only one pigment while others produced several; Flavobacterium arbo-
rescens contained five distinct hydrocarbon carotenoids. Some species which
can be separated by fermentation reactions produced identical pigment.
Twelve strains of Staphylococcus aureus, obtained from various sources, were
subjected to pigment analysis. Pigments whose absorption maxima were
identicalwiththoseof delta-carotene and rubixanthinwere found in all the strains
studied. The strain used as a test organism for determining phenol coefficients

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also contained another hydrocarbon. Four strains isolated from food poisoning
cases and four from staphylococcic infections also contained an ester of a carot-
enol with absorption maxima the same as those of rubixanthin.
It is apparent that bacteria offer interesting possibilities for discovering the
function of the carotenoid pigments in nature.
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