HYDROCARBON DISTRIBUTION OF ALGAE AND BACTERIA, AND MICROBIOLOGICAL ACTIVITY IN SEDIMENTS* - PNAS
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HYDROCARBON DISTRIBUTION OF ALGAE AND BACTERIA,
AND MICROBIOLOGICAL ACTIVITY IN SEDIMENTS*
BY JERRY HAN AND MELVIN CALVIN
DEPARTMENT OF CHEMISTRY, UNIVERSITY OF CALIFORNIA, 'BERKELEY
Communicated June 27, 1969
Abstract.-The chemical taxonomic relationship of microorganisms has been
studied through the hydrocarbon fraction of their chemical constituents. The
diagenesis and biological transformations of some hydrocarbons in sediments is
suggested, as a result of this information.
Various compounds of presumed biological origin have been isolated from
petroleum,' ancient sediments,2-4 and meteorites.5 Among these classes of
organic compounds, the hydrocarbons have probably received the most atten-
tion. Meinscheinr found that the extracts of soil contained more odd than even
carbon-numbered normal paraffins. Bray' reported that the ratio of odd over
even carbon-numbered n-paraffins is significant in Recent sediments, approxi-
mately 2.4 to 5.5. However, the value decreases for older sediments, and it is
close to 1.0 in petroleum. The isoprenoid hydrocarbons with the regular head-
tail-head-tail linkage have been taken to be the residue of life forms.3
Barghoorn and his co-workers8' 9 claimed to have found blue-green algae and
bacterialike microfossils in Precambrian rocks. It is very important to obtain
confirmatory evidence for the existence of these primitive microorganisms in the
Precambrian period.
Chemical criteria can be used as confirmatory evidence for classifying living
organisms which were previously identified solely on the basis of morphologic
characteristics. Chemistry may have more to contribute than any morpholog-
ical analysis, not only because of the relative evanescence of most plant tissues in
geological deposits, but also because the biochemistry of evolutionary processes
may be deduced from the presence of compounds from known diagenic pathways.
The evolutionary step from the procaryotic cell (blue-green algae and bac-
teria) to the eucaryotic cell (green algae, fungi, protozoa, higher plants, and
animals) is recognized by the appearance (or presence, in some cases) of nuclear
membrane, mitotic division, chromosome number, cytoplasmic streaming, and
mitochondria in the eucaryotic cell. Since lipids are important constituents of
cytoplasmic and intracellular membranes, chemical taxonomic studies have
been made to determine whether such evolutionary transitions are reflected at
the molecular level.
We have analyzed the hydrocarbon constituents of four species of blue-green
algae (Table 1), seven species of nonphotosynthetic bacteria (Table 2), and six
species of green algae and photosynthetic bacteria (Table 3). The method used
to extract, fractionate, and analyze the hydrocarbons from the algae and bacteria
has been described in our preliminary report.10
The freeze-dried cells were sonicated with 150 ml 3: 1 benzene: methanol by
stirring for 30 minutes. Then the total sample and supernatant were transferred
436
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TABLE 1. Hydrocarbons from algae.
Nostoc Anacy8tis Phormidium Chorogloea ChloreUa
muscorum'1 nidulans" luridum" fritschii12 pyrenoidosall
(blue-green) (blue-green) (blue-green) (blue-green) (green algae)
n-C15 0.35 20.60 ... ... 0.13
n-C16 0.35 2.50 ... 0.26 0.073
A-C17 ... 2.95 ... ... 81.5
n-C17 82.75 73.75 96.00 87.30 18.0
7- and 8-methyl-
heptadecane 16.10 0.15 4.00 0.09 ...
4-methyiheptadecane ... ... ... 12.20 ...
n-C,8 0.41 ... ... 0.09 0.055
TABLE 2. Hydrocarbons from nonphotosynthetic bacteria.
Micro- C18- C08- Desulfo-
P. shermanii coccus tridium tridium Desulfo- vibrio
E. coli (anaero- lysodei- tetano- acidi- vibrio Hilden-
(aerobic)1I bic) 5 kiticus81 morphum'5 urici16 Essex 616 borough16
n-C,5 0.5 2.1 8.7 1.6 1.0 0.3 0.3
n-C16 1.7 2.6 8.0 2.1 14.4 0.3 0.3
Pristane ... 46.5 2.5 3.5 2.1 0.5 0.7
n-C17 5.5 13.3 8.1 9.1 50.0 1.5 2.3
Phytane ... 1.0 1.9 2.5 1.3 0.3 0.3
n-C18 27.6 3.6 5.0 7.2 4.5 2.7 3.3
n-C1, 12.0 3.8 12.5 7.4 4.9 16.3 10.4
n-C20 10.0 3.8 5.0 5.5 3.1 34.0 16.5
n-C21 5.5 4.2 2.9 4.7 1.9 26.4 11.3
n-C22 6.0 4.1 0.9 5.1 1.7 9.2 5.0
n-C23 8.3 3.1 0.5 6.7 1.0 2.9 3.2
n-C24 7.4 1.5 0.4 7.1 1.0 1.3 4.6
n-C21 6.0 1.0 0.1 5.8 0.7 0.7 7.9
n-C26 3.3 0.5 ... 5.0 0.5 0.7 13.3
n-C27 3.3 0.5 ... 4.3 ... 0.5 10.8
n-C28 0.5 ... ... 0.9 ... 0.5 5.0
n-C29 1.4 ... ... ... ... ...
n-C30 .. ... ... ... ... ... 1.
n-Cal ... ... ... ... ... ... 0.5
to a soxhlet apparatus and extracted with benzene: methanol for eight hours.
After solvent was removed from the extract on a rotary evaporator, the organic
residue was separated into three groups by column chromatography. The
column contained 100 gm activated alumina which had been washed with 150
ml n-heptane. The residue of the original extract was placed on top of the
column. The first fraction containing the aliphatic hydrocarbons was eluted
from the column with n-heptane, the second with benzene, and the third with
methanol. In this report the composition of only the first fractions (hydro-
carbons) will be discussed. After most of the solvent was removed from the
n-heptane fraction, the sample was then analyzed by capillary gas chromatog-
raphy and mass spectrometry. All mass spectra were taken using a combina-
tion of an Aerograph 204 gas chromatograph and an AEI MS-12 mass spectrom-
eter. The gas chromatographic oven temperature was programmed from 900
to 3000C at 2°C/minute with a helium flow rate of 2.5 ml/minute. The effluent
from the capillary column was split into two parts, 1.5 ml/minute going to the
flame ionization detector and 1 ml/minute going through a 1 ft X 0.002 inch i.d.
heated stainless steel tube at 2200C into the ion source of the mass spectrometer.
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TABLE 3. Photosyntheticc bacteria.
Rho&-- fRhodopsacdo- Rhodomicro-
8pirillum nwnas Chlorobrium bium
rubrumI sl)heroidcsll (sulfurbacteria) 17 vannieliil4
n-C,5 0.01 0.87 1.5 0.03
n-C,' 0.06 3.1 0.75 0.05
Pristane 0.10 9.6 0.5 0.14
A-C17 * . . . . .
n-C,7 3.50 42.5 50.0 0.15
Phytane 1.3 0.5 0.16
n-CI8 0.35 19.2 1.3 0.37
n-Cig 0.45 18.8 1.3 0.45
n-C20 0.32 0.38 1.0 0.46
n-C21 0.24 1.5 0.46
n-C22 .. . 3.0 0.62
n-C23 *. .
. .
4.1 1.0
n-C24 ... 6.9 1.2
n-CM, 10.8 0.92
n-C26 13.1 0.50
n-C27 2.1 0.38
n-C28 0.5 0.15
Squalene and high-mol-
wt cyclic hydrocarbons 94.7 92.5
Normal paraffins are among the most stable of all biogenic organic compounds
and are thought to be diagnostic of biologically produced organic matter which
can be derived from the decarboxylation of fatty acids. The normal heptadecane
is the dominant compound in the hydrocarbon fraction of all the photosynthetic
microorganisms, but the predominance is not found in aerobic and anaerobic
nonphotosynthetic bacteria. Johns et al.4 reported that the n-C17 alkane was a
major peak in the total normal alkane fraction of the Soudan Shale (2.5 X 109
years). This may indicate that photosynthetic microorganisms are a major
hydrocarbon source in the Soudan Shale.
Normal alkanes of carbon number less than C14 and more than Cu are rarely
present to any appreciable extent in blue-green algae and green algae (Table 1).
However, the hydrocarbons from photosynthetic and nonphotosynthetic bacteria
range in chain length from C13 to C31 (Tables 2 and 3). These hydrocarbon dis-
tributions are very different from the higher plants which contain normal hydro-
carbons ranging from C23 to C35.18 The difference between the normal hydro-
carbon patterns of microorganisms and higher plants is striking, and there ap-
pears to be a future for taxonomic correlation based on this approach.
In the Recent lake sediment, the Mud Lake of Florida (5000 years),'0 n-C29,
and n-C31 alkanes are the most important peaks in the total hydrocarbon frac-
tion. The Green River Formation (50 X 106 years) has two peak maxima in the
normal alkane fraction,4 one at n-C,7 and another one at n-C29 and n-C31. It
appears that the high molecular weight of odd-numbered paraffins, n-C27, n-C29,
and n-C31 are contributed by higher plants. In the Green River Formation the
higher plants and photosynthetic microorganisms appear to play equally im-
portant roles in hydrocarbon production.
If we disregard the n-C17 hydrocarbons, the amounts of odd-numbered ho-
mologs and the even-numbered ones in either algae or bacteria are about equal
(Tables 1, 2, and 3), in contrast to higher plants.'8 Since the alkanes can be
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assumed to be end products of the living organism metabolism,'9 this may be
significant in terms of the specificity of the enzyme systems which are involved
in the formation of the alkanes from acetate units. It is probable that one route
to the living orgaiuism hydrocai'hons involves decarboxylation of the correspond-
ing long-chain fatty acids. The decarboxylation of the fatty acids of chain
length CI0-C34 which might give rise to the typical n-alkanes are dependent on
the enzyme specificity of the organisms.
The 50:50 mixture of 7- and 8-methylheptadecane'0 appeared to occur uniquely
in the blue-green algae and was absent in other photosynthetic and nonphoto-
synthetic bacteria. These hydrocarbons have also not been found in the green
algae, which are more advanced than blue-green algae. These methyl-branched
alkanes seemed to be of particular significance because the methyl branches are
not the iso- and anteiso-structures which have been found in many living orga-
nisms. This pair of hydrocarbons may be of considerable importance in
taxonomic studies, not only because of the large amount found in the blue-green
algae, but also because of its unique presence in such a primitive microorganism.
The biological occurrence and structures of this branched C18 hydrocarbon
has been confirmed previously.20 Some evidence indicates'9 that the biosyn-
thesis of this mixture involves vaccenic acid (cis-11-octadecenoic acid). A
methyl group is added to the double bond of vaccenic acid, perhaps via the cyclo-
propane intermediate. This is followed by decarboxylation to yield the 7-
and 8-methylheptadecanes.
The next step is to find out whether there is any evidence that this peculiar
structure is present in Precambrian rock itself. Since blue-green algae fossils
are believed to be present in some of the ancient rocks,8 9 the branched-C18
hydrocarbon becomes an extremely important biological marker to prove the
occurrence of primitive algae in that age. The locations of the branched-C1s
hydrocarbon coincided with peak a in Figure 1. This implies that blue-green
algae were probably present at the time of the formation of the Soudan Shale.
The isoprenoid hydrocarbons are absent in blue-green algae and green algae,
but they are widely distributed in most of the photosynthetic and nonphoto-
synthetic bacteria. The highest content of pristane was found in P. shermanji
(46.5%) (Fig. 2), an anaerobic nonphotosynthetic bacteria. This implies the
anaerobic nonphotosynthetic bacteria has the enzymatic system which produces
the isoprenoid hydrocarbons although the chlorophylls are absent. This in-
dicates that the nonphotosynthetic anaerobic bacterial activity may play an
important role in isoprenoid hydrocarbon diagenesis.
It has been suggested8 9 that in the early period of Precambrian time no living
organism existed on the earth except procaryotic cells, bacteria, and blue-green
algae. Probably after the blue-green algae began to decline, bacteria became
more active. The isoprenoid hydrocarbons, higher-molecular-weight hydro-
carbons, and those unresolved branched and cyclic hydrocarbons were produced
by anaerobic nonphotosynthetic bacteria.
Figure 3, the total hydrocarbons from Clostridium tetanomorphum H-i, shows
the distribution of normal alkanes and more than a hundred resolved peaks be-
tween n-C,6 and n-C26. Mass spectra indicate these unresolved peaks are
Downloaded by guest on January 29, 2022440 CHEMISTRY: HAN AND CALVIN PROC. N. A. S.
n-CJ n-C18
njn-C7
SOUDAN ~~Pristanel
Apiezon L Phytane
90I1-2500 n - C16
2 C /min.r
2.5 mlI/min. H e
a n-2
I ~~~n-CI5
b-C18
SOU DAN
TOTAL H/C
with coinj. of b-C18
FIG. 1.-The top figure shows the gas chromatogram of total aliphatic hydrocarbons from
extractable organic material of the Soudan Shale. The bottom figure shows this fraction with
coinjection of branched-C18 hydrocarbons. All the conditions are described in the Figure.
branched and cyclic hydrocarbons, which also existed in every Precambrian
rock.
As shown in Table 3, some of the photosynthetic bacteria, Rhodospirillum
rubrum and Rhodomicrobium vannielii, contain approximately 92-95 per cent
branched and cyclic hydrocarbons in the total hydrocarbon mixture. Those
have a gas chromatographic retention time between n-Cs and n-C32. The mass
spectroscopic data indicates the predominant peak in both cases is squalene.
Approximately 1 per cent of the steranes and tr-iterpanes can also be detected
by gas chromatography mass spectrometry. The individual structures of these
Downloaded by guest on January 29, 2022VOL. 64, 1969 CHEMISTRY: HAN AND CALVIN 441
FIG. 2.-Gas chromatogram of total aliphatic hydrocarbons from extractable organic material
of P. shermanii.
FIG. 3.-Gas chromatogram of total aliphatic hydrocarbons from extractable organic material
of Clostridium tetanmorphum H-i.
cyclic hydrocarbons are still under investigation. These high-molecular-weight
branched and cyclic compounds are not present in any significant amount in any
other procaryotic cells.
The abundance of the steranes and triterpanes is a striking feature of the total
Downloaded by guest on January 29, 2022442 CHEMISTRY: HAN AND CALVIN PROC. N. A. S.
Co
"0 hydrocarbon content of the Green River
Shale,4 but they are only present in the
0 ~
.0 Nonesuch and Soudan Shales in very small
OQ
amoun ts.2 It is probable that one route
to the high-molecular-weight cyclic hydro-
0o
~~~4. carbons involves some reactions of the
2 $n i
4
sterols. The sterols which may give rise
X¢
CO to the typical steranes and triterpanes by
geological processes are common constit-
c
uents of eucaryotic cells. It is suggested
.0 that the initial reaction process has to take
a) place by the reaction of the functional
groups of sterols. This could then be fol-
lowed by reduction, isomerization, ther-
8 CC 0 00
vv0 mal cracking, as well as anaerobic bacterial
o5
"-
0 C4 q L4
000
activities during geological time to give
z
VV
q.j a)
en
high-molecular-weight cyclic hydrocar-
Co
C bons in the Green River Shale.
The use of chemical constituents and
¢ a) so-called molecular characteristics as an
.0 .0 .0 -.0
. aid to the classification of living organ-
isms is not only a familiar concept now,
but also a very useful tool. These charac-
0
teristics have the advantage over morpho-
Co4 f00
C.
0 Cs
w
0
.0A logical ones in that they can be very ex-
AA
.0
actly described in terms of definite chemi-
cal structures. The elucidation of the
II:$
0
structures and configurations of secondary
o Ca) c:a) organic products can lead to an under-
C.)e C0-2
m
C-
0 standing not only of their biosyntheses,
Vad *
0l but of possible subsequent diagenetic
.0 changes as well. Erdtman2' has pointed
a)o out that the most valuable substances
0f
taxonomically are not those which are in-
Ca C volved in primary metabolic processes but
c; C ,C_S
)
0
c;
rather those which are relatively stable
secondary products. The hydrocarbons
C
o+
0) c)
meet this requirement and also represent
the most easily analyzed chemical sub-
o
CoC stances in living organisms. The distribu-
tion of hydrocarbons ranging from Clo to
.^ ., C35 has been discussed previously in this
report and the generalized results are
shown in Table 4. The chemical taxo-
nomic features are summarized as follows:
(a) The mixture of 7- and 8-methyl
Downloaded by guest on January 29, 2022VOL. 64, 1969 CHEMISTRY: HAN AND CALVIN 443
heptadecane is only present in blue-green algae and absent in any other living
organism so far examined.
(b) The isoprenoid hydrocarbons, pristane and phytane, are absent in algae
and aerobic bacteria but are generally present in the others.
(c) Squalene, steranes, and triterpanes are absent in algae and nonphoto-
synthetic bacteria but are present in most cases in photosynthetic bacteria and
higher plants.
(d) In all cases, the odd over even carbon-numbered n-paraffins is approxi-
mately 1.0 in aerobic and anaerobic bacteria, while it is greater than 10 in higher
plants. The value is 1.0 to 5.0 in algae when the predominant component n-C17
is excluded.
(e) The n-C17 hydrocarbon is predominant in algae, the dominance is de-
creased in photosynthetic bacteria. The intensity is not outstanding in non-
photosynthetic bacteria and generally absent in higher plants.
U) The n-C27, n-C29, and n-C3O alkanes are the major constituents in higher
plants, within the normal distribution range in bacteria, but they are absent in
algae.
We appreciate the generosity of Dr. N. G. Carr, J. R. Postgate, and H. A. Barker, for
providing samples.
* This work was supported in part by the U.S. Atomic Energy Commission and in part by
the National Aeronautics and Space Administration.
1 Blumer, M., Science, 149, 722 (1965).
2 Meinschein, W. G., Bull. Am. Assoc. Petrol. Geologists, 43, 925 (1959).
3 Eglinton, G., and M. Calvin, Scientific American, 216, 32 (1967).
4 Johns, R. B., T. Belsky, E. D. McCarthy, A. L. Burlingame, P. Haug, H. K. Schnoes, W.
Richter, and M. Calvin, Geochim. Cosmochim. Acta, 30, 1191 (1966).
5 Hayes, J. M., Geochim. Cosmochim. Ada, 31, 1395 (1967).
6 Meinschein, W. G., and G. S. Kenny, Anal. Chem., 29, 1153 (1957).
7 Bray, E. E., and E. D. Evans, Geochim. Cosmochim. Ada, 22, 2 (1961).
8 Schopf, J. W., and E. S. Barghoorn, Science, 156, 508 (1967).
9 Barghoorn, E. S., and J. W. Schopf, Science, 152, 758 (1966).
10 Han, J., E. D. McCarthy, W. Van Hoeven, M. Calvin, and W. H. Bradley, these PRo-
CEEDINGS, 59, 29 (1968).
11 Which were grown in inorganic medium under light, 25-30'C, with air: CO2(95:5). They
were cultured in this laboratory.
12 Sample was supplied by Dr. N. G. Carr, University of Liverpool. It was grown in in-
organic medium with light and C02.
13 Sample was purchased from Miles Laboratories, Inc., Elkhart, Ind.
14 Sample was obtained from Dr. N. G. Carr, University of Liverpool. R. vanielii was grown
on a malate-glutamate medium, anaerobically in the light.
16 Samples were supplied by Dr. H. A. Barker, Department of Biochemistry, University of
California, Berkeley.
16 Cells were obtained from Dr. J. R. Postgate, University of Sussex. They were cultured
in sodium lactate medium.
17 These specimens were grown in inorganic sulfur medium under light without air.
18Eglinton, G., and R. J. Hamilton, Chemical Plant Taxonomy (London and New York:
Academic Press, 1963), chap. 8.
19 Han, J., H. Chan, and M. Calvin, J. Amer. Chem. Soc., 91, 5156 (1969).
20 Han, J., E. D. McCarthy, M. Calvin, and M. H. Benn, J. Chem. Soc. (C), 2785 (1968).
21 Erdtman, H., in Perspectives in Organic Chcmistry, ed. A. Todd (New York: Interscience,
1956), p. 473.
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